Immunology and Immuno-Technology

WU, Nekemte
May, 2014
Desalegn Amenu (M.Sc.) Wollega University, 2014
Page i
Immunology and Immuno-technology 2014
Table of contents
Table of contents ............................................................................................................................... ii
List of tables ...................................................................................................................................... v
Preface .............................................................................................................................................. ix
1. Historical Background of Immunology ......................................................................................... 1
1.
2.
3.
Innate (Nonspecific) Immune Response .................................................................................... 2

1.1.
Overview of the immune system ........................................................................................ 2
1.2.
Innate host defenses against infection: Anatomical barriers ............................................... 4
1.3.
Innate host defenses against infection: Humoral barriers ................................................... 5
1.4.
Innate host defenses against infection: Cellular barriers .................................................... 6
1.5.
Phagocyte response to infection ......................................................................................... 6
1.6.
Non-specific killer cells ..................................................................................................... 9
1.7.
Determinants recognized by the innate immune response .................................................. 9
Complement............................................................................................................................. 10
2.1.
Complement Functions .................................................................................................... 10
2.2.
Pathways of Complement Activation ............................................................................... 11
2.2.1.
Classical Pathway ..................................................................................................... 12
2.2.2.
Lectin Pathway ......................................................................................................... 15
2.2.3.
Alternative Pathway ................................................................................................. 16
2.3.
Membrane Attack (Lytic) Pathway .................................................................................. 20
2.4.
Biologically Active Products of Complement Activation ................................................ 21
Antigens ................................................................................................................................... 22
3.1.
Factors influencing immunogenicity ................................................................................ 22
3.2.
Chemical nature of immunogens ...................................................................................... 23
3.3.
Types of antigens ............................................................................................................. 23
3.4.
Antigenic determinants recognized by B cells and Ab ..................................................... 24
3.5.
Determinants recognized by T cells ................................................................................. 25
3.6.
Superantigens ................................................................................................................... 25
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4.
Immunoglobulins: Structure and Function ............................................................................... 27

4.1.
General Functions of Immunoglobulins ........................................................................... 27
4.2.
Basic Structure of Immunoglobulins ................................................................................ 28
5.5.
Structure of the Variable Region ...................................................................................... 29
5.6.
Immunoglobulin Fragments: Structure/Function Relationships ....................................... 30
5.7. Human Immunoglobulin Classes, Subclasses, Types and Subtypes ..................................... 32

5.8.
Structure and Some Properties of Ig Classes and Subclasses ........................................... 33
6. Immunoglobulins: Isotypes, Allotypes and Idiotypes .................................................................. 37

7. Immunoglobulins: Genetics........................................................................................................ 41
8. Antibody Formation .................................................................................................................... 50
8.1. General Characteristics of the Antibody Response................................................................... 50
8.2. Antibody Formation.............................................................................................................. 51
8.3. Kinetics of antibody responses to T-dependent Ag .............................................................. 52
8.4. Specificity of 1o and 2o responses ....................................................................................... 53
8.5. Qualitative changes in Ab during 1o and 2o responses........................................................ 54
8.6. Cellular events during 1o and 2o responses to T-dependent Ag .......................................... 56
9. Immunization............................................................................................................................... 59
10. Cells of the Immune System and Antigen Recognition ............................................................. 64
11. Major Histocompatibility Complex and T Cell Receptors ........................................................ 71
12. Antigen Process and Presentation .............................................................................................. 79
13. Cell-Cell Interactions in Immune Responses............................................................................. 86
14. Cytokines ................................................................................................................................... 96
15. Immunoregulation ................................................................................................................... 105
16. MHC: genetics and role in transplantation .............................................................................. 108
17. Tolerance and Autoimmunity................................................................................................... 124
17.1. Tolerance Introduction:..................................................................................................... 124
17.2. Autoimmunity ................................................................................................................... 127
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18. Tumor Immunology ................................................................................................................. 130

19. Immunodeficiency ................................................................................................................... 136
20. Antibody and Antigen Reaction .............................................................................................. 140
21. Serological Techniques............................................................................................................ 148
22. Human Immunodeficiency Virus (HIV) .................................................................................. 169
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List of tables
Table 1: Complete protein ............................................................................................................... 11
Table 2: Biological Activity of classical pathway products............................................................. 15
Table 3: Regulation of the Classical Pathway ................................................................................ 15
Table 4: CD Marker cell................................................................................................................. 67
Table 5: types of cytokines .............................................................................................................. 91
Table 6: Immunoglobulin regulation ............................................................................................. 105
Table 7: Factors which determine induction of immune response or tolerance following challenge
with antigen. .................................................................................................................................. 127
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List of figures
Figure 1: overview of immune system .............................................................................................. 3
Figure 2: Cells of Immune system..................................................................................................... 4
Figure 3: phagocytic cells.................................................................................................................. 7
Figure 4: Phagocytosis ...................................................................................................................... 7
Figure 5: Nitric oxide-dependent killing ........................................................................................... 9
Figure 6: nitric oxide toxic ................................................................................................................ 9
Figure 7: PAMP and PRR ............................................................................................................... 10
Figure 8: Path way of complement activation ................................................................................. 12
Figure 9: Generation of C3 convertase in the classical pathway ..................................................... 13
Figure 10: Generation of C5convertase in the classical pathway .................................................... 14
Figure 11: Generation of C3 convertase in the lectin pathway ........................................................ 16
Figure 12: Generation of C5 convertase in the lectin pathway ........................................................ 17
Figure 13: Spontaneous activation of C3......................................................................................... 18
Figure 14: Regulation of activated C3 by DAF ............................................................................... 19
Figure 15: Regulation by CR1, Factor H and Factor I..................................................................... 20
Figure 16: Stabilized C3 convertase of the alternative pathway ...................................................... 21
Figure 17: antigenic determinants.................................................................................................... 25
Figure 18: T cell Receptor ............................................................................................................. 26
Figure 19: Immunoglobulin ............................................................................................................. 27
Figure 20: Basic structure of immunoglobulin (Heavy and Light chain) ....................................... 28
Figure 21: variable index of Ig ........................................................................................................ 29
Figure 22: Immunoglobulin fragments ............................................................................................ 30
Figure 23: Antigen Binding and FC receptor .................................................................................. 31
Figure 24: F(ab) 2 fragments......................................................................................................... 31
Figure 25: Immunoglobulin classes ................................................................................................. 32
Figure 26: Immunoglobulin J Chain and C4 ................................................................................ 34
Figure 27: Immunoglobulin Tail piece ........................................................................................... 35
Figure 28: Immunoglobulin structure and ................................................................................ 35
Figure 29: Immunoglobulin secretory piece .................................................................................... 36
Figure 30: IgE Tail piece ................................................................................................................ 36
Figure 31: IgE C4 ......................................................................................................................... 36
Figure 32: Ig Isotopes Kappa .......................................................................................................... 37
Figure 33: Ig Allotypes.................................................................................................................... 38
Figure 34: Ig Idiotypes location....................................................................................................... 41
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Figure 35: Light chain gene families ............................................................................................... 42

Figure 36: Gene rearrangement and Expression ............................................................................... 43
Figure 37: Heavy chain gene family................................................................................................ 44
Figure 38: VDJ arrangement ........................................................................................................... 45
Figure 39: VDJ Transcription .......................................................................................................... 45
Figure 40: Mechanism of DNA rearrangements .............................................................................. 46
Figure 41: Ig genes are expressed in a B cell (Heavy chain) ........................................................... 47
Figure 42: Ig genes are expressed in a B cell (light chain) .............................................................. 48
Figure 43: antibody formation ......................................................................................................... 51
0
0
Figure 44: antibody formation after immunization (1 and (2 ) ....................................................... 53
Figure 46 : antibody affinity ............................................................................................................ 54
0
Figure 47: affinity maturation (1 ) ................................................................................................... 55
0
Figure 48: affinity maturation (2 ) ................................................................................................... 55

Figure 49: Cellular events during 1o ............................................................................................... 56
Figure 50: Cellular events during 2o responses .............................................................................. 56
Figure 51: Cellelular events during secondary response.................................................................. 57
Figure 52.class switching ................................................................................................................ 58
Figure 53: polyadenylation sites ...................................................................................................... 59
Figure 54: types of immunity .......................................................................................................... 60
Figure 55: Newer adjuvant formulations ......................................................................................... 63
Figure 56: stem cell of immune system ........................................................................................... 67
Figure 57: B cell and T cell ............................................................................................................ 68
Figure 58: B and T cell origination ................................................................................................. 70
Figure 59: lymphatic lymphocytes .................................................................................................. 71
Figure 60: B and T cell differentiation ............................................................................................ 71
Figure 61: Structure of MHC class I ............................................................................................... 73
Figure 62: Structure of MHC class II .............................................................................................. 74
Figure 63: Role of TCR in the immune response ............................................................................ 75
Figure 64: TCR a heterodimer ......................................................................................................... 76
Figure 65: TCR chains ............................................................................................................... 77
Figure 66: Antigen presenting cell .................................................................................................. 77
Figure 67: TCR and CD3 Recognition ............................................................................................ 77
Figure 68: MHC class I pathway ..................................................................................................... 81
Figure 69: MHC class II pathway.................................................................................................... 82
Figure 70: random VDJ recombination ........................................................................................... 85
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Figure 71: TCP selection ................................................................................................................. 86

Figure 72: Central role of Th cells in immune response.................................................................. 87
Figure 73: Cytokine production ....................................................................................................... 88
Figure 74: B cell proliferation ......................................................................................................... 90
Figure 75: Cell-cell interactions in primary Ab response ................................................................ 91
Figure 76: Process of Antigen presentation ..................................................................................... 93
Figure 77: Fas- and TNF-mediated killing ..................................................................................... 94
Figure 78: Granule-mediated killing ............................................................................................... 94
Figure 79: cytokine production, bactericidal and tumoricidal activities ......................................... 95
Figure 80: Receptors for cytokines are heterodimers (A) ................................................................ 97
Figure 81: Receptors for cytokines are heterodimers (B) ................................................................ 98
Figure 82: Interferon ..................................................................................................................... 100
Figure 83: activate NK cells and monocytes, proliferative phase................................................. 101
Figure 85: Cytokine networks ....................................................................................................... 104
Figure 86: Immunoregulation by antibody ................................................................................... 106
Figure 87: The human MHC gene complex................................................................................... 109
Figure 88: The mouse MHC complex ........................................................................................... 111
Figure 89: Co-dominant expression of MHC antigens ................................................................... 112
Figure 90: Activation of CTL and Mechanism of Allograft Destruction ....................................... 114
Figure 92: types of hypersensitivity reaction ................................................................................. 121
Figure 93: Mechanism of damage in type-III hypersensitivity ...................................................... 122
Figure 94: Mechanisms of damage in delayed hypersensitivity .................................................... 123
Figure 95: Latex agglutination reaction ........................................................................................ 143
Page viii
Preface
Immunology and immunotechnology is an advanced science dealing with how the human immune
system organized, function and the different types of serological techniques are applied. It is a very
vast subject covering a wide area of technology. This teaching material is prepared based on the
existing curriculum of immunology and immunotechnology and consists of different chapters with
different subtopics. Therefore, the material is designed to present clear and concise understanding
about Immunology and immunotechnology; and it is primarily suitable for students following
Bachelor and Master programme in Biology, Medical and any Microbiology. Finally, it is quite
obvious that it had demanded a lot of effort in preparing this material. However, it should be noted
that even then, there could be constructive comments which are helpful in improving this lecture
note. Thus, it will be well accepted and acknowledged for the contribution.
Page ix
1.. Historical Background of Immunology

Immunology is defined as the study of the molecules, cells, organs, and systems responsible for the
recognition and disposal of foreign material. Immunology began as a branch of microbiology. The
study of infectious disease and the bodys response to them has a major role for the development of
immunology. Moreover, the concept of germ theory of disease has contributed to the field of
immunology.
It was Edward Jenner who first studied the response of the body to foreign substances. He
observed that dairy maids who had naturally contracted a mild infection called cowpox seemed to
be protected against smallpox, a horribly disfiguring disease and a major killer. In 1796, Jenner
inoculated an eight year-old boy with fluid from cowpox blisters on the hand of a dairymaid. The
boy contracted cowpox. Then two month later Jenner inoculated him with fluid from a small pox
blister, the boy only developed a small sore at the site of inoculation. His exposure to the mild
disease cowpox had made him immune to the small pox infection. These were some of the vital
events occurred in the history of immunology following Jenners achievement. In 1879, the first
human pathogen, gonococcus, was isolated by Neisser. In 1883, Klebs and Loeffler isolated
diphtheria bacilli which led to the production of the first defined antigen, diphtheria toxin, by Roux
and Yersin in 1888. In the same year the first antibodies, serum bactericidins, were reported by
Nuttal and Pasteur.
In 1890, von Behring and Kitasato discovered antitoxins that led to the development of toxoids for
diphtheria and tetanus. In 1900, Land Steiner discovered the blood group antigens and their
corresponding antibodies. This led to the ability to give blood transfusion without provoking
reactions. It was in 1916 that the first journal of immunology began publication in which many of
new findings published on it. In general, immunology has always depended on and stimulated the
application of technology, such as the use of microscopy, electrophoresis,
immunoelectrofluorescence, etc. Thus Immunology and Serology immunology has not become an
inborn discipline but has maintained close associations with many other fields of medical sciences.
Immunology is the study of our protection from foreign macromolecules or invading organisms
and our responses to them. These invaders include viruses, bacteria, protozoa or even larger
parasites. In addition, we develop immune responses against our own proteins (and other
molecules) in autoimmunity and against our own aberrant cells in tumor immunity.
Desalegn Amenu (M.Sc, Microbiology)
Page 1
Our first line of defense against foreign organisms is barrier tissues such as the skin that stop the
entry of organism into our bodies. If, however, these barrier layers are penetrated, the body
contains cells that respond rapidly to the presence of the invader. These cells include macrophages
and neutrophils that engulf foreign organisms and kill them without the need for antibodies.
Immediate challenge also comes from soluble molecules that deprive the invading organism of
essential nutrients (such as iron) and from certain molecules that are found on the surfaces of
epithelia, in secretions (such as tears and saliva) and in the blood stream. This form of immunity is
the innate or non-specific immune system that is continually ready to respond to invasion.
A second line of defense is the specific or adaptive immune system which may take days to
respond to a primary invasion (that is infection by an organism that has not hitherto been seen). In
the specific immune system, we see the production of antibodies (soluble proteins that bind to
foreign antigens) and cell-mediated responses in which specific cells recognize foreign pathogens
and destroy them. In the case of viruses or tumors, this response is also vital to the recognition and
destruction of virally-infected or tumorigenic cells. The response to a second round of infection is
often more rapid than to the primary infection because of the activation of memory B and T cells.
We shall see how cells of the immune system interact with one another by a variety of signal
molecules so that a coordinated response may be mounted. These signals may be proteins such as
lymphokines which are produced by cells of the lymphoid system, cytokines and chemokines that
are produced by other cells in an immune response, and which stimulate cells of the immune
system.
1. Innate (Nonspec
(Nonspecific)
ecific) Immune Resp
Response
1.1. Overview of the immune system
We are constantly being exposed to infectious agents and yet, in most cases, we are able to
resist these infections. It is our immune system that enables us to resist infections. The
immune system is composed of two major subdivisions, the innate or non-specific immune system
and the adaptive or specific immune system (Figure 1). The innate immune system is our first
line of defense against invading organisms while the adaptive immune system acts as a second
line of defense and also affords protection against re- exposure to the same pathogen. Each of
the major subdivisions of the immune system has both cellular and humoral components by
which they carry out their protective function (Figure 1). In addition, the innate immune system
also has anatomical features that function as barriers to infection. Although these two arms of
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the immune system have distinct functions, there is interplay between these systems (i.e.,
components of the innate immune system influence the adaptive immune system and vice versa).
There are two phases to the immune response: pathogen recognition and pathogen
removal. Although the innate and adaptive immune systems both function to protect against
invading organisms, they differ in a number of ways. The adaptive immune system requires
some time to react to an invading organism, whereas the innate immune system includes defenses
that, for the most part, are constitutively present and ready to be mobilized upon infection.
Second, the adaptive immune system is antigen specific and reacts only with the organism that
induced the response. In contrast, the innate system is not antigen specific and reacts equally
well to a variety of organisms. Finally, the adaptive immune system demonstrates
immunological memory. It remembers that it has encountered an invading organism and
reacts more rapidly on subsequent exposure to the same organism. In contrast, the innate immune
system does not demonstrate immunological memory.
Figure 1: overview of immune system
All cells of the immune system have their origin in the bone marrow. They include myeloid
(neutrophils, basophils, eosinophils, macrophages, and dendritic cells) and lymphoid cells (B
lymphocytes, T lymphocytes, and natural killer cells) (Figure 2).
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Immunology and Immuno-technology

Immuno technology 2014
Figure 2: Cells of Immune system

The main function of the immune system is self/non-self discrimination.
nation. This ability to
distinguish between self and non-self is necessary to protect the organism from invading
pathogens and to eliminate modified or altered cells (e.g.
(
malignant cells).
). Since pathogens may
replicate intracellular (virusses and some bacteria and parasites) or extracellular (most bacteria,
fungi and parasites), different components
co
of the immune system have evolved to protect against
these different types of pathogens. It is important to remember that infecti
ection with an organism
does not necessarily mean diseases, since in most cases the immune system will be able to
eliminate the infection before
ore disease occurs. Disease occurs only when the bolus of infection is
high, when the virulence of the invading organism is great or when imm
munity is compromised.
Although the immune system,
syste for the most part, has beneficial effects,
s, there can be detrimental
detri
effects as well. During inflammation, which is the response to an invading organism,
organis there may be
local discomfort
fort and collateral damage
da
to healthy
hy tissue as a result of the toxic products produced
by the immune response. In addition, in some cases the immune response can be directed
toward self tissues resulting in autoimmune disease.
1.2. Innate host defenses against iinnfection: Anatom
Anatomical barriers
1) Mechanical factors: epithelial
ithelial surfaces
s
form a physical barrier that is very impermeable
i
to most
infectious agents. Thus, the skin acts as our first line of defense against invading organis
organisms. The
desquamation of skin epithelium
epitheli
also helps remove bacteria and other infectious
ectious agents that have
adhered to the epithelial surffaces. Movement due to cilia or peristalsis helps to keep air passages
and the gastrointestinal tractt free from microorganisms. The flushing action of tears and saliva
Page 4
helps prevent infection of the eyes and mouth.
The trapping affect of mucus that lines the
respiratory and gastrointestinal tract helps protect the lungs and digestive systems from infection.
2) Chem
Lysozyme and
Chemical factors
factors: fatty acids in sweat inhibit the growth of bacteria.
phospholipase found in tears, saliva and nasal secretions can breakdown the cell wall of bacteria
and destabilize bacterial membranes. The low pH of sweat and gastric secretions prevents growth
of bacteria. Defensins (low molecular weight proteins) found in the lung and gastrointestinal
tract have antimicrobial activity. Surfactants in the lung act as opsonins (substances that promote
phagocytosis of particles by phagocytic cells).
3)
Biological factors: the normal flora of the skin and in the gastrointestinal tract can
prevent the colonization of pathogenic bacteria by secreting toxic substances or by competing with
pathogenic bacteria for nutrients or attachment to cell surfaces.
1.3. Innate host defenses against inf

infectio
tion: Hum
Humoral barriers
i.
Anatom
Anato
mical barriers are very effective in preventing colonization of tissues by microorganisms.
However, when there is damage to tissues the anatomical barriers are breached and infection may
occur. Once infectious agents have penetrated tissues, another innate defense mechanism
comes into play, namely acute inflammation. Humoral factors play an important role in
inflammation, which is characterized by edema and the recruitment of phagocytic cells. These
ii.
humoral factors are found in serum or they are formed at the site of infection.
compleement syste
The compl
system is the major humoral non-specific defense mechanism. Once activated
complement can lead to increased vascular permeability, recruitment of phagocytic cells, and lysis
and opsonization of bacteria.
iii.
Depending on the severity of the tissue injury, the coagulation system may or may not be
activated. Some products of the coagulation system can contribute to the non-specific defenses
because of their ability to increase vascular permeability and act as chemotactic agents for
phagocytic cells. In addition, some of the products of the coagulation system are directly
antimicrobial. For example, beta-lysin, a protein produced by platelets during coagulation
iv.
v.
vi.
can lyse many Gram positive bacteria by acting as a cationic detergent.

By binding iron,
iron an essential nutrient for bacteria, lactoferrin and transferrin limit bacterial
growth.
Lyso
Lysozyme breaks down the cell wall of bacteria.
Cytoki
kines
Cyto
ki
nes have various effects depending on the balance.
Interferons are proteins that can
limit virus replication in cells. Some interleukins induce fever and the production of acute phase
Page 5
proteins, some of which are antimicrobial because they can opsonize bacteria.
1.4. Innate host defenses against inf
infectio
tion: Cellu
Cellular barriers
1. Part of the inflammatory response is the recruitment of polymorphonuclear eosinophils and
macrophages to sites of infection. These cells are the main line of defense in the non-specific
immune system.
2. Neutrophils, Polymorphonuclear cells (PMNs), are recruited to the site of infection where
they phagocytose invading organisms and kill them intracellularly.
In addition, PMNs
contribute to collateral tissue damage that occurs during inflammation.
3. Tissue macrophages and newly recruited monocytes, which differentiate into macrophages, also
function in phagocytosis and intracellular killing of microorganisms. In addition, macrophages
are capable of extracellular killing of infected or altered self target cells.
Furthermore,
macrophages contribute to tissue repair and act as antigen- presenting cells, which are required
for the induction of specific immune responses.
4. Natural killer (NK) and lymphokine activated killer (LAK) cells can nonspecifically kill virus
infected and tumor cells. These cells are not part of the inflammatory response but they are
important in nonspecific immunity to viral infections and tumor surveillance.
ffective
rtaain parasites.
5. Eosinophils have proteins in granules that are eff
ective in killing cceert
1.5. Phagocyte response to infection
i.
Circulating PMNs and monocytes respond to danger (SOS) signals generated at the site of an
infection. SOS signals include N-formyl-methionine containing peptides released by bacteria,
clotting system peptides, complement products and cytokines released from tissue macrophages
that have encountered bacteria in tissue. Some of the SOS signals stimulate endothelial cells
near the site of the infection to express cell adhesion molecules such as ICAM-1 and selectins
which bind to components on the surface of phagocytic cells and cause the phagocytes to adhere
to the endothelium. Vasodilators produced at the site of infection cause the junctions between
endothelial cells to loosen and the phagocytes then cross the endothelial barrier by squeezing
between the endothelial cells in a process called diapedesis (Figure 3). Once in the tissue spaces
some of the SOS signals attract phagocytes to the infection site by chemotaxis (movement toward
an increasing chemical gradient). The SOS signals also activate the phagocytes, which results in
increased phagocytosis and intracellular killing of the invading organisms.
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Figure 3: phagocytic cells
ii.
Phagocytosis: Phagocytic cells have a variety of receptors on their cell membranes through
which infectious agents bind to the cells (Figure 4A).
These include Fc receptors,
complement receptors, scavenger receptors, and toll-like receptors.
After attachment of a
bacterium, the phagocyte begins to extend pseudopods around the bacterium (Figure 4B). The
pseudopods eventually surround the bacterium and engulf it, and the bacterium is enclosed in a
phagosome (Figure 4C). During phagocytosis the granules or lysosomes of the phagocyte
fuse with the phagosome and empty their contents (Figure 4D). The result is a bacterium
engulfed in a phagolysosome which contains the contents of the granules or lysosomes.
Figure 4: Phagocytosis
iii.
Respiratory burst.
burst During phagocytosis there is an increase in glucose and oxygen consumption
which is referred to as the respiratory burst. The consequence of the respiratory burst is that a
number of oxygen-containing compounds are produced which kill the bacteria being
phagocytosed.
This is referred to as oxygen-dependent intracellular killing. In addition,
bacteria can be killed by pre-formed substances released from granules or lysosomes when they
fuse with the phagosome. This is referred to as oxygen-independent intracellular killing.
Page 7
a) OxygenOxygen-dependent myeloperoxidas
yeloperoxidase (MPO)-independent intracellular killing. During
phagocytosis glucose is metabolized via the pentose monophosphate shunt and NADPH is
formed. Cytochrome B which was part of the granule combines with the plasma membrane
NADPH oxidase and activates it. The activated NADPH oxidase uses oxygen to oxidize the
NADPH. The result is the production of superoxide anion. Some of the superoxide anion is
converted to H2O2 and singlet oxygen by superoxide dismutase. In addition, superoxide anion
can react with H2O2 resulting in the formation of hydroxyl radical and more singlet oxygen. The
result of all of these reactions is the production of the toxic oxygen compounds superoxide
anion (O2-), H2O2, singlet oxygen (1O2) and hydroxyl radical (OH).
b) OxygenOxygen-dependent MPO-dependent intracellu
intracellular killing. As the azurophilic granules fuse with
the phagosome, myeloperoxidase is released into the phagolysosome. MPO utilizes H2O2 and
halide ions (usually Cl-) to produce hypochlorite, a highly toxic substance. Some of the
hypochlorite can spontaneously break down to yield singlet oxygen. The result of these reactions
is the production of toxic hypochlorite (OCl-) and singlet oxygen (1O2).
c) Detoxification reactions.
reactions PMNs and macrophages have means to protect themselves from the toxic
oxygen intermediates. These reactions involve the disputation of superoxide anion to hydrogen
peroxide by superoxide dismutase and the conversion of hydrogen peroxide to water by catalase.
d) OxygenOxygen-ind
independent intracellular killing.
In addition to the oxygen-dependent mechanisms of
killing there are also oxygenindependent killing mechanisms in phagocytes: cationic proteins
(cathepsin) released into the phagolysosome can damage bacterial membranes; lysozyme
breaks down bacterial cell walls; lactoferrin chelates iron, which deprives bacteria of this required
nutrient; hydrolytic enzymes break down bacterial proteins. Thus, even patients who have defects
in the oxygen- dependent killing pathways are able to kill bacteria. However, since the oxygendependent mechanisms are much more efficient in killing, patients with defects in these pathways
are more susceptible and get more serious infections.
e) Nitric oxideoxide-dependent killing.
killing Binding of bacteria to macrophages, particularly binding via Tolllike receptors, results in the production of TNF-alpha, which acts in an autocrine manner to induce
the expression of the inducible nitric oxide synthetase gene (i-nos ) resulting in the production of
nitric oxide (NO). If the cell is also exposed to interferon gamma (IFN-gamma) additional nitric
oxide will be produced. Nitric oxide released by the cell is toxic and can kill microorganism in
the vicinity of the macrophage (Figure 6).
Page 8

dependent killing
Figure 5: Nitric oxide-dependent
Figure 6:: nitric oxide toxic
1.6. NonNon-specific killer cells
a) Several different cells including NK cells, activated macrophages, eosinophils, and mast cells are
capable of killing foreign and altered self target cells in a non-specific manner.
m
These play an
important role in the innate
nate immune
im
system.
b) Innate response to virus infection
ection and altered self (transformed cells): NK
K cells have two kinds of
receptors on their surface, NK receptor and inhibitory receptor (Figure 7). When the NK receptor
encounters its ligand on a taarget cell, thee NK cell is signaled to kill. However, if the inhibitory
receptor also binds its ligand (MHC class I) then the killing signal is reppressed. Normal cells
constitutively express MHC
C class I on their surface,
e, however virus infected and transformed
tra
cells down regulate expression
xpression of MHC class I. Thus, NK cells
ells selectively kill virus-infected and
transformed cells while sparing
ring normal
nor
cells.
c) Innate response to extracell
extracelluular microorganis
croorganism
ms (parasites): eosinophils are a specialized group of
cells with the ability to engage and damage
d mage large extracellular parasites, such as schistosomes.
schistoso
Activated eosinophils release
relea
their granule components including major basic protein,
eosinophil peroxidase (a cationic hemoprotein), and eosinophil cationic protein (a
ribonuclease that is an eosinophil-specific
eosinophil
toxin that is very potent at killing many parasites).
Determ
imm
1.7. Deter
minants recognized by the innate im
mune rreesponse
a) Determinants
inants recognized by components of the innate (nonspecific)
(
) immune system differ from
those recognized by the adaptive (specific) immune system. Antibodies and the B and T cell
receptors recognize discrete
te determinants
d
and demonstrate
onstrate a high degree of specificity, enabling
the adaptive immune system to recognize and react to a particular pathogen. In contrast,
components of the innate immune system recognize broad molecular patterns found in
pathogens but not in the host. Thus, they lack a high degree of specificity seen in the adaptive
immune system. The broad molecular patterns recognized by the innate immune system have
Page 9

been called PAMPS (pathogen associated molecular patterns) and the receptors for PAMPS are
called PRRs (pattern recognition receptors).
rece
A particular PRR can recognize a molecular pattern
that may be present on a number
nu ber of different pathogens enabling the receptor to recognize a
variety of different pathogens. Exa
Examples of some PAMPs and
nd PRRs are illustrated
illustr
in Figure 8.
Figure 7:: PAMP and PRR

2. Complement
2.1. Complement Functions
Historically, the term complement (C) was used to refer to a heat-labile
heat labile serum component that was
able to lyse bacteria (activity is destroyed (inactivated) by heating serum at 56 degrees C for 30
minutes). However, complement is now known to contribute to host defenses in other ways as
well. Complement can opsonize bacteria for enhanced phagocytosis; it can recruit and activate
various cells including polymorphonuclear cells (PMNs) and macrophages; it can participate in
regulation of antibody responses and it can aid in the clearance of immune complexes and
apoptotic cells. Complement can also have detrimental effects for the host; it contributes to
inflammation and tissue damage and it can trigger anaphylaxis.
Complement comprises over 20 different serum proteins (see Table 1) that are produced by a
variety of cells including, hepatocytes, macrophages and gut epithelial cells. Some complement
proteins bind to immunoglobulins or to membrane components of cells. Others are proenzymes
Page 10
that, when activated, cleave one or more other complement proteins. Upon cleavage some of the
complement proteins yield fragments that activate cells, increase vascular permeability or opsonize
bacteria.
Table 1: Complete protein
C1(qrs), C2, C3, C4, C5. C6, C7, C8 and C9
Factors B, D, H, I and Properdin (P)
Mannose binding lectin (MBL), MBL-associated serine proteases (MASP-1 and MASP-2)
C1 inhibitor (C1-INH, serpin), C4-binding protein (C4-BP), decay accelerating factor (DAF)
Complement receptor 1 (CR1) protein S (vitronectin)
2.2. Pathways of Complement Activation
Complement activation can be divided into four pathways (figure 1): the classical pathway, the
lectin pathway, the alternative pathway and the membrane attack (or lytic) pathway. Both classical
and alternative pathways lead to the activation of C5 convertase and result in the production of
C5b which is essential for the activation of the membrane attack pathway.
Page 11
Figure 8: Path way of complement activation
2.2.1. Classical Pathway

C1 activation
C1, a multi-subunit protein containing three different proteins (C1q, C1r and C1s), binds to the Fc
region of IgG and IgM antibody molecules that have interacted with antigen. C1 binding does not
occur to antibodies that have not completed with antigen and binding requires calcium and
magnesium ions. (N.B. In some cases C1 can bind to aggregated immunoglobulin [e.g. aggregated
IgG] or to certain pathogen surfaces in the absence of antibody). The binding of C1 to antibody is
via C1q and C1q must cross link at least two antibody molecules before it is firmly fixed. The
binding of C1q results in the activation of C1r which in turn activates C1s. The result is the
formation of an activated C1qrs, which is an enzyme that cleaves C4 into two fragments C4a
and C4b.
Page 12
Figure 9: Generation of C3 convertase in the classical pathway
C4 and C2 activation (generation of C3 convertase).

The C4b fragment binds to the membrane and the C4a fragment is released into the
microenvironment. Activated C1qrs also cleaves C2 into C2a and C2b. C2a binds to the
membrane in association with C4b, and C2b is released into the microenvironment. The resulting
C4bC2a complex is a C3 convertase, which cleaves C3 into C3a and C3b.
Page 13
Figure 10: Generation of C5convertase in the classical pathway
C3 activation (generation of C5 convertase)

C3b binds to the membrane in association with C4b and C2a, and C3a is released into the
microenvironment. The resulting C4bC2aC3b is a C5 convertase. The generation of C5 convertase
is the end of the classical pathway. Several of the products of the classical pathway have potent
biological activities that contribute to host defenses. Some of these products may also have
detrimental effects if produced in an unregulated manner. Table 2 summarizes the biological
activities of classical pathway components. If the classical pathway were not regulated there would
be continued production of C2b, C3a, and C4a. Thus, there must be some way to regulate the
activity of the classical pathway. Table 3 summarizes the ways in which the classical pathway is
regulated. The importance of C1-INH in regulating the classical pathway is demonstrated by the
result of a deficiency in this inhibitor. C1-INH deficiencies are associated with the development of
hereditary angioedema.
Page 14
Table 2: Biological Activity of classical pathway products.

Component
C2b
C3a
C3b
C4a
C4b
Biological Activity
Prokinin; cleaved by plasmin to yield kinin, which results in edema
Anaphylotoxin; can activate basophils and mast cells to degranulate resulting
in increased vascular permeability and contraction of smooth muscle cells,
which may lead to anaphylaxis
Opsonin; promotes phagocytosis by binding to complement receptors
Activation of phagocytic cells
Anaphylotoxin (weaker than C3a)
Opsonin; promotes phagocytosis by binding to complement receptors
Table 3: Regulation of the Classical Pathway

Compone
Regulation
nt
All
C1-INH; dissociates C1r and C1s from C1q
C3a
C3a inactivator (C3a-INA;Carboxypeptidase B); inactivates C3a
C3b
Factors H and I; Factor H facilitates the degradation of C3b by Factor I
C4a
C3-INA
C4b
C4 binding protein(C4-BP) and Factor I; C4-BP facilitates

degradation of C4b by Factor I; C4-BP also prevents association of C2a with C4b thus
blocking the formation of C3 convertase
2.2.2. Lectin Pathway

The lectin pathway (Figure 4) is very similar to the classical pathway. It is initiated by the binding
of mannose-binding lectin (MBL) to bacterial surfaces with mannose-containing polysaccharides
(mannans). Binding of MBL to a pathogen results in the association of two serine proteases,
MASP-1 and MASP-2 (MBL-associated serine proteases). MASP-1 and MASP-2 are similar to
C1r and C1s, respectively and MBL is similar to C1q. Formation of the MBL/MASP-1/MASP-2
tri-molecular complex results in the activation of the MASPs and subsequent cleavage of C4 into
C4a and C4b. The C4b fragment binds to the membrane and the C4a fragment is released into the
Page 15
microenvironment. Activated MASPs also cleave C2 into C2a and C2b. C2a binds to the
membrane in association with C4b and C2b is released into the microenvironment. The resulting
C4bC2a complex is a C3 convertase, which cleaves C3 into C3a and C3b. C3b binds to the
membrane in association with C4b and C2a and C3a is released into the microenvironment. The
resulting C4bC2aC3b is a C5 convertase. The generation of C5 convertase is the end of the lectin
pathway. The biological activities and the regulatory proteins of the lectin pathway are the same as
those of the classical pathway.
Figure 11: Generation of C3 convertase in the lectin pathway

2.2.3. Alternative Pathway
The alternative pathway begins with the activation of C3 and requires Factors B and D and Mg++
cation, all present in normal serum.
1. Amplification loop of C3b formation
In serum there is low level spontaneous hydrolysis of C3 to produce C3i. Factor B binds to C3i and
becomes susceptible to Factor D, which cleaves Factor B into Bb. The C3iBb complex acts as a C3
convertase and cleaves C3 into C3a and C3b. Once C3b is formed, Factor B will bind to it and
becomes susceptible to cleavage by Factor D. The resulting C3bBb complex is a C3 convertase
that will continue to generate more C3b, thus amplifying C3b production. If this process continues
unchecked, the result would be the consumption of all C3 in the serum. Thus, the spontaneous
production of C3b is tightly controlled.
Page 16
Figure 12: Generation of C5 convertase in the lectin pathway

2. Control of the amplification loop
As spontaneously produced C3b binds to autologous host membranes, it interacts with DAF (decay
accelerating factor), which blocks the association of Factor B with C3b thereby preventing the
formation of additional C3 convertase. In addition, DAF accelerates the dissociation of Bb from
C3b in C3 convertase that has already formed, thereby stopping the production of additional C3b.
Some cells possess complement receptor 1 (CR1). Binding of C3b to CR1 facilitates the enzymatic
degradation of C3b by Factor I. In addition, binding of C3 convertase (C3bBb) to CR1 also
dissociates Bb from the complex. Thus, in cells possessing complement receptors, CR1 also plays a
role in controlling the amplification loop. Finally, Factor H can bind to C3b bound to a cell or in
the in the fluid phase and facilitate the enzymatic degradation of C3b by Factor I. Thus, the
amplification loop is controlled by either blocking the formation of C3 convertase, dissociating C3
convertase, or by enzymatically digesting C3b. The importance of controlling this amplification
loop is illustrated in patients with genetic deficiencies of Factor H or I. These patients have a C3
deficiency and increased susceptibility to certain infections.
Page 17
Figure 13: Spontaneous activation of C3

3.. Stabilization of C convertase by activator (protector) surfaces
When bound to an appropriate activator of the alternative pathway, C3b will bind Factor B, which
is enzymatically cleaved by Factor D to produce C3 convertase (C3bBb). However, C3b is
resistant to degradation by Factor I and the C3 convertase is not rapidly degraded, since it is
stabilized by the activator surface. The complex is further stabilized by properdin binding to
C3bBb. Activators of the alternate pathway are components on the surface of pathogens and
include: LPS of Gram-negative bacteria and the cell walls of some bacteria and yeasts. Thus, when
C3b binds to an activator surface, the C3 convertase formed will be stable and continue to generate
additional C3a and C3b by cleavage of C3.
Page 18
Figure 14: Regulation of activated C3 by DAF

4. Generation of C5 convertase
Some of the C3b generated by the stabilized C3 convertase on the activator surface associates with
the C3bBb complex to form a C3bBbC3b complex. This is the C5 convertase of the alternative
pathway. The generation of C5 convertase is the end of the alternative pathway. The alternative
pathway can be activated by many Gram-negative (most significantly, Neisseria meningitidis and
N. gonorrhoea), some Gram-positive bacteria and certain viruses and parasites, and results in the
lysis of these organisms. Thus, the alternative pathway of C activation provides another means of
protection against certain pathogens before an antibody response is mounted. A deficiency of C3
results in an increased susceptibility to these organisms. The alternate pathway may be the more
primitive pathway and the classical and lectin pathways probably developed from it.
Remember that the alternative pathway provides a means of non-specific resistance against
infection without the participation of antibodies and hence provides a first line of defense against a
number
of
infectious
agents.
Many gram negative and some gram positive bacteria, certain viruses, parasites, heterologous red
cells, aggregated immunoglobulins (particularly, IgA) and some other proteins (e.g. proteases,
clotting pathway products) can activate the alternative pathway. One protein, cobra venom factor
(CVF), has been extensively studied for its ability to activate this pathway.
Page 19
Figure 15: Regulation by CR1, Factor H and Factor I

2.3. Membrane Attack (Lytic) Pathway
C5 convertase from the classical (C4b2a3b), lectin (C4b2a3b) or alternative (C3bBb3b) pathway
cleaves C5 into C5a and C5b. C5a remains in the fluid phase and the C5b rapidly associates with
C6 and C7 and inserts into the membrane. Subsequently C8 binds, followed by several molecules
of C9. The C9 molecules form a pore in the membrane through which the cellular contents leak
and lysis occurs. Lysis is not an enzymatic process; it is thought to be due to physical damage to
the membrane. The complex consisting of C5bC6C7C8C9 is referred to as the membrane attack
complex (MAC).
C5a generated in the lytic pathway has several potent biological activities. It is the most potent
anaphylotoxin. In addition, it is a chemotactic factor for neutrophils and stimulates the respiratory
burst in them and it stimulates inflammatory cytokine production by macrophages. Its activities are
controlled by inactivation by carboxypeptidase B (C3-INA). Some of the C5b67 complex formed
can dissociate from the membrane and enter the fluid phase. If this were to occur it could then bind
to other nearby cells and lead to their lysis. The damage to bystander cells is prevented by Protein
S (vitronectin). Protein S binds to soluble C5b67 and prevents its binding to other cells.
Page 20
Figure 16: Stabilized C3 convertase of the alternative pathway
2.4. Biologically Active Products of Complement Activation

Activation of complement results in the production of several biologically active molecules which
contribute to resistance, anaphylaxis and inflammation.
Kinin production.
C2b generated during the classical pathway of C activation is a prokinin which becomes
biologically active following enzymatic alteration by plasmin. Excess C2b production is prevented
by limiting C2 activation by C1 inhibitor (C1-INH) also known as serpin which displaces C1rs
from the C1qrs complex (Figure 16). A genetic deficiency of C1-INH results in an overproduction
of C2b and is the cause of hereditary angioneurotic edema. This condition can be treated with
Danazol which promotes C1-INH production or with -amino caproic acid which decreases
plasmin activity.
Anaphylotoxins
C4a, C3a and C5a (in increasing order of activity) are all anaphylotoxins which cause
basophil/mast cell degranulation and smooth muscle contraction. Undesirable effects of these
peptides are controlled by carboxypeptidase B (C3a-INA).
Chemotactic Factors
Page 21
C5a and MAC (C5b67) are both chemotactic. C5a is also a potent activator of neutrophils,
basophils and macrophages and causes induction of adhesion molecules on vascular endothelial
cells.
Opsonins
C3b and C4b in the surface of microorganisms attach to C-receptor (CR1) on phagocytic cells and
promote phagocytosis. Other Biologically active products of C activation Degradation products of
C3 (iC3b, C3d and C3e) also bind to different cells by distinct receptors and modulate their
functions.
In summary, the complement system takes part in both specific and non-specific resistance and
generates a number of products of biological and pathophysiological significance (Table 4). There
are known genetic deficiencies of most individual C complement components, but C3 deficiency is
most serious and fatal. Complement deficiencies also occur in immune complex diseases (e.g.,
SLE) and acute and chronic bacterial, viral and parasitic infections.
3. Antigens
1) Definitions
2) Immunogen - a substance that induces a specific immune response.
3) Antigen (Ag) - a substance that reacts with the products of a specific immune response.
4) Hapten - a substance that is nonimmunogenic but which can react with the products of a specific
immune response. Haptens are small molecules which could never induce an immune response
when administered by themselves but which can when coupled to a carrier molecule. Free haptens,
however, can react with products of the immune response after such products have been elicited.
Haptens have the property o Antigenicity but not immunogenicity.
5) Epitope or Antigenic Determinant - the portion of an antigen that combines with the products
of a specific immune response. Antibody (Ab) - a specific protein which is produced in response
to an immunogen and which reacts with an antigen.
3.1. Factors influencing imm
immunogenicit
ogenicity
a) Contribution of the immunogen: The immune system normally discriminates between self and
non-self such that only foreign molecules are immunogenic. There is not absolute size above
which a substance will be immunogenic. However, in general, the larger the molecule the more
immunogenic it is likely to be. In general, the more complex the substance is chemically the
more immunogenic it will be. The antigenic determinants are created by the primary sequence of
residues in the polymer and/or by the secondary, tertiary or quaternary structure of the molecule.
The physical form, such as whether it is particulate or soluble can affect immunogenicity.
In
Page 22
general, particulate antigens are more immunogenic than soluble ones and denatured antigens
more immunogenic than the native form. Antigens that are easily degradable and phagocytosed
are generally more immunogenic. This is because for most antigens (T-dependant antigens, see
below) the development of an immune response requires that the antigen be phagocytosed,
processed and presented to helper T cells by an antigen presenting cell (APC)
b) Contribution of the Biological System: Genetic factors of the host can determine immunogenicity
of an antigen. Some substances are immunogenic in one species but not in another. Similarly, some
substances are immunogenic in one individual but not in others (i.e. responders and nonresponders). The species or individuals may lack or have altered genes that code for the receptors
for antigen on B cells and T cells or they may not have the appropriate genes needed for the APC
to present antigen to the helper T cells. Age can also influence immunogenicity. Usually the very
young and the very old have a diminished ability to mount an immune response in response to an
immunogen.
c) Method of Administration: The dose of administration of an immunogen can influence its
immunogenicity. There is a dose of antigen above or below which the immune response will not be
optimal. For example, high doses of antigen can often be tolerogenic and blunt the immune
response. Generally the subcutaneous route is for effective for inducing an immune response than
the intravenous or intragastric routes. The route of antigen administration can also alter the nature
of the response. Substances that can enhance the immune response to an immunogen are called
adjuvants. The use of adjuvants, however, is often hampered by undesirable side effects such as
fever and inflammation.
3.2. Chemical nature of immunogens
a) The vast majority of immunogens are proteins. These may be pure proteins or they may be
glycoproteins or lipoproteins. In general, proteins are usually very good immunogens. Pure
polysaccharides and lipopolysaccharides are good immunogens. Nucleic acids are usually poorly
immunogenic. However they may become immunogenic when single stranded or when complexed
with proteins. In general lipids are non-immunogenic, although they may be haptens. Some
glycolipids and phospholipids can stimulate T cells and produce a cell-mediated immune response
3.3. Types of antigens
a) T-independent antigens are antigens which can directly stimulate the B cells to produce antibody
without the requirement for T cell help. In general, polysaccharides are T- independent antigens.
The responses to these antigens differ from the responses to other antigens. These antigens are
characterized by the same antigenic determinant repeated many times. Many of these antigens
Page 23
can activate B cell clones specific for other antigens (polyclonal activation).
T-independent
antigens can be subdivided into Type 1 and Type 2 based on their ability to polyclonally activate
B cells. Type 1 T-independent antigens are polyclonal activators while Type 2 are not. Tindependent antigens are generally more resistant to degradation and thus they persist for longer
periods of time and continue to stimulate the immune system.
b) T-dependent antigens are those that do not directly stimulate the production of antibody without
the help of T cells. Proteins are T-dependent antigens. Structurally these antigens are
characterized by a few copies of many different antigenic determinants.
c) Hapten-carrier conjugates are immunogenic molecules to which haptens have been covalently
attached. The immunogenic molecule is called the carrier. Structurally these conjugates are
characterized by having native antigenic determinants of the carrier as well as new
determinants created by the hapten (haptenic determinants). The actual determinant created by
the hapten consists of the hapten and a few of the adjacent residues, although the antibody
produced to the determinant will also react with free hapten. In such conjugates the type of
carrier determines whether the response will be T-independent or T-dependent.
3.4. Antigenic determinants recognized by B cells and Ab
Antigenic determinants recognized by B cells and the antibodies secreted by B cells are created by
the primary sequence of residues in the polymer (linear or sequence determinants) and/or by the
secondary, tertiary or quaternary structure of the molecule (conformational determinants). In
general antigenic determinants are small and are limited to 4-8 residues. Although, in theory, each
4-8 residues can constitute a separate antigenic determinant, in limited to approximately 4-8
residues. (Amino acids and or sugars). The combining site of f an antibody will accommodate an
antigenic determinant of the number of antigenic determinants per antigen is much lower than what
would theoretically be possible. Usually the antigenic determinants are limited to those portions of
the antigen that are accessible to antibodies as illustrated in the Figure 17 (antigenic determinants
are indicated in black).
Page 24

Figure 17: antigenic determinants

3.5. Determ
Determinants recognized by T cells
a) Antigenic determinants
inants recognized by T cells are created by the primary sequence of
amino
ino acids in proteins. T cells do not recognize polysaccharide or nucleic acid antigens. This is
why polysaccharides are generally T-independent antigens and proteins are generally T-dependent
T
antigens. The determinants
inants need nott be located on the exposed surface of the antigen since
recognition of the determinant
inant by T cells requires that the antigen b e p r o t e o l y t i c a l l y
d e g r a d e d into s m a l l e r p e p t i d e s . Free p e p t i d e s a r e not recognized by T cells, rather
the peptides associate with molecules coded for by the major Histocompatibility
Histoco
complex
(MHC) and it is the complex
mplex of MHC molecules + peptide that is recognized by T cells. Some T
cells can recognize lipids in conjunction with a MHC-like molecule caalled CD1. In general
antigenic determinants are sm
mall and are limited to approximately 8-15 amino
ino acids. Although, in
theory, each 8-15
15 residues can constitute a separate antigenic determinant,
ant, in practice, the number
of antigenic determinants per antigen is much less than what would theoretically be possible.
The antigenic determinants are limited to those portions of the antigen that can bind to MHC
molecules. This is why there can be differences in the responses
ses of different individuals.
3.6. Superantigens
a) When the immune system encounters a conv
conventional T-dependent
dependent antigen, only a small
s
fraction
(1 in 104 -105 ) of the T cell population is able to recognize thee antigen and become
activated (monoclonal/oligoclonal
onoclonal/oligoclonal response).
However, there are so
some antigens which
polyclonally activate a large
rge fraction of the T cells (up to 25%). These antigens are called
superantigens (Figure 18). Examples
Ex
of superantigens include: Staphylococcal enterotoxins (food
Page 25

poisoning), Staphylococcal toxic shock toxin (toxic shock syndrome),

e), Staphylococcal exfoliating
toxins (scalded skin syndrome)
syndro
and Streptococcal pyrogenic exotoxins (shock). Although the
bacterial superantigens are the best studied there are superantigens associated with viruses and
other microorganisms as well. The diseases associated with exposure to superantigens are, in
part, due to hyper activation of the immune system and subsequent release of biologically active
cytokines by activated T cells.
Figure 18: T cell

cell Receptor
Page 26
4. Immunoglobulins: Structure and Function

1. Definition
Immunoglobulins (Ig) Glycoprotein molecules which are produced by plasma cells in response to
an immunogen and which function as antibodies. The immunoglobulins derive their name from
the finding that when antibody-containing serum
is placing in an electrical
fields the
antibodies, which were responsible for immunity, migrated with the globular proteins (Figure 19).
Figure 19: Immunoglobulin

4.1. General Functions of Immunoglobulins
Ag binding - Immunoglobulins bind specifically to one or a few closely related antigens. Each
immunoglobulin actually binds to a specific antigenic determinant. Antigen binding by antibodies
is the primary function of antibodies and can result in protection of the host.
Valency. The valence of antibody refers to the number of antigenic determinants that an individual
antibody molecule can bind. The valence of all antibodies is at least two and in some instances
more.
Effector Functions - Often the binding of an antibody to an antigen has no direct biological effect.
Rather, the significant biological effects are a consequence of secondary "effector functions" of
antibodies. The immunoglobulins mediate a variety of these effector functions. Usually the
ability to carry out a particular effector function requires that the antibody bind to its antigen. Not
every immunoglobulin will mediate all effector functions. Fixation of complement - lysis of cells,
release of biologically active molecules Binding to various cell types - phagocytic cells,
lymphocytes, platelets, mast cells, and basophils have receptors that bind immunoglobulins and
the binding can activate the cells to perform some function. Some immunoglobulins also bind to
Page 27
receptors on placental trophoblasts. The binding results in transfer of the immunoglobulin across
the placenta and the transferred maternal antibodies provide immunity to the fetus and newborn
4.2. Basic Structure of Immunoglobulins
The basic structure of the immunoglobulins is illustrated in the Figure 2. Although different
immunoglobulins can differ structurally they all are built from the same basic unit.
A.
Heavy and Light Chains - All immunoglobulins have a four chain structure as their basic unit. They
are composed of two identical light chains (23Kd) and two identical heavy chains (50-70Kd).
Figure 20: Basic structure of immunoglobulin (Heavy and Light chain)

B.
Disulfide bonds
1. Inter-chain - The heavy and light chains and the two heavy chains are held together by interchain disulfide bonds and by non-covalent interactions. The number of interchain disulfide bonds
varies among different immunoglobulin molecules.
Intra-chain - Within each of the polypeptide chains there are also intra-chain disulfide bonds.
C. Variable (V) and Constant (C) Regions - After the amino acid sequences of many different
heavy chains and light chains were compared, it became clear that both the heavy and light chain
could be divided into two regions based on variability in the amino acid sequences.
1. Light Chain - VL (110 aa) and CL (110 aa)
2. Heavy Chain - VH (110 aa) and CH (330-440 aa)
D. Hinge Region - The region at which the arms of the antibody molecule forms a Y is called the
hinge region because there is some flexibility in the molecule at this point.
Page 28
E. Domains - 3D images of the immunoglobulin molecule shows that it is not straight as depicted
in Figure 2. Rather, it is folded into globular regions each of which contains an intra-chain
disulfide bond. These regions are called domains.
1. Light Chain Domains - VL and CL
2. Heavy Chain Domains - VH, CH1 - CH3 (or CH4)
F. Oligosaccharides - Carbohydrates are attached to the CH2 domain in most immunoglobulins.
However, in some cases carbohydrates may also be attached at other locations.
Figure 21: variable index of Ig

5.5.
Structure of the Variable Region

A. Hypervariable (HVR) or complementarity determining regions (CDR)
Comparisons of the amino acid sequences of the variable regions of Ig's show that most of the
variability resides in three regions called the hypervariable regions or the complementarity
determining regions as illustrated in Figure 3. Antibodies with different specificities (i.e. different
combining sites) have different CDR's while antibodies of the exact same specificity have
identical CDR's (i.e. CDR --> Ab Combing site). CDR's are found in both the H and the L chains.
B. Framework regions
The regions between the CDR's in the variable region are called the framework regions (FR)
(Figure 22). Based on similarities and differences in the framework regions the immunoglobulin
heavy and light chain variable regions can be divided into groups and subgroups. These represent
the products of different variable region genes.
Page 29
5.6.
Immunoglobulin Fragments: Structure/Function Relationships

Immunoglobulin fragments produced by proteolytic digestion have proven very useful in
elucidating structure/function relationships in immunoglobulins.
A. Fab - Digestion with papain breaks the immunoglobulin molecule in the hinge region before
the H-H inter-chain disulfide bond Figure 4. This results in the formation of two identical
fragments that contain the light chain and the VH and CH1 domains of the heavy chain.
1. Antigen binding - These fragments were called the Fab fragments because they contained the
antigen binding sites of the antibody. Each Fab fragment is monovalent whereas the original
molecule was divalent. The combining site of the antibody is created by both VH and VL. An
antibody is able to bind a particular antigenic determinant because it has a particular combination
of VH and VL. Different combinations of a VH and VL result in antibodies that can bind
different antigenic determinants.
Figure 22: Immunoglobulin fragments

B.Fc - Digestion with papain also produces a fragment that contains the remainder of the two
heavy chains each containing a CH2 and CH3 domain. This fragment was called Fc because it
was easily crystallized. Effector functions - The effector functions of immunoglobulins are
mediated by this part of the molecule. Different functions are mediated by the different domains
in this fragment (See Figure 5). Normally the ability of an antibody to carry out an effector
function requires the prior binding of an antigen. However, there are exceptions to this rule.
Page 30
Figure 23: Antigen Binding and FC receptor
Figure 24: F(ab) 2 fragments
C.
F(ab')2 - Treatment of immunoglobulins with pepsin results in cleavage of the heavy chain after
the H-H inter-chain disulfide bonds resulting in a fragment that contains both antigen binding sites
(Figure 23). This fragment was called F (ab') 2 because it was divalent. The Fc region of the
molecule is digested into small peptides by pepsin. The F(ab')2 binds antigen but it does not
Page 31
mediate the effector functions of antibodies.

5.7.. Human Immunoglobulin Classes, Subclasses, Types and Subtypes
A.Immunoglobulin classes - The immunoglobulins can be divided into 5 different classes based on
differences in the amino acid sequences in the constant region of the heavy chains. All
immunoglobulins within a given class will have very similar heavy chain constant regions. These
differences can be detected by sequence studies or more commonly by serological means (i.e. by
the use of antibodies directed to these differences).
1. IgG - Gamma () heavy chains
2. IgM - Mu () heavy chains
3. IgA - Alpha () heavy chains
4. IgD - Delta () heavy chains
5. IgE - Epsilon () heavy chains
Figure 25: Immunoglobulin classes

B.Immunoglobulin Subclasses - The classes of immunoglobulins can be divided into subclasses
based on small differences in the amino acid sequences in the constant region of the heavy chains.
All immunoglobulins within a subclass will have very similar heavy chain constant region amino
acid sequences. Again these differences are most commonly detected by serological means.
1. IgG Subclasses
a)
IgG1 - Gamma 1 (1) heavy chains b)
IgG2 - Gamma 2 (2) heavy chains c)
IgG3 -
Gamma 3 (3) heavy chains d) IgG4 - Gamma 4 (4) heavy chains

2. IgA Subclasses
a) IgA1 - Alpha 1 (1) Heavy chains b) IgA2 - Alpha 2 (2) heavy chains
C.
Immunoglobulin Types - Immunoglobulins can also be classified by the type of light chain that
they have. Light chain types are based on differences in the amino acid sequence in the
constant region of the light chain. These differences are detected by serological means.
Page 32
1. Kappa light chains ()

2. Lambda light chains ()
D.Immunoglobulin Subtypes - The light chains can also be divided into subtypes based on differences
in the amino acid sequences in the constant region of the light chain.
1. Lambda subtypes a)
Lambda 1
(1)
b) Lambda 2 (2)
c) Lambda 3 (3)
d) Lambda 4 (4)
E.
Nomenclature - Immunoglobulins are named based on the class, or subclass of the heavy chain
and type or subtype of light chain. Unless it is stated precisely you are to assume that all subclass,
types and subtypes are present. IgG means that all subclasses and types are present.
F. Heterogeneity - Immunoglobulins considered as a population of molecules are normally very
heterogeneous because they are composed of different classes and subclasses each of which has
different types and subtypes of light chains. In addition, different immunoglobulin molecules
can have different antigen binding properties because of different VH and VL regions.
5.8.
Structure and Some Properties of Ig Classes and Subclasses

A
IgG
1. Structure - The structures of the IgG subclasses are presented in Figure 7. All IgG's are monomers
(7S immunoglobulin). The subclasses differ in the number of disulfide bonds and length of the
hinge region.
2. Properties - Most versatile immunoglobulin because it is capable of carrying out all of the
functions of immunoglobulin molecules.
1) IgG is the major Ig in serum - 75% of serum Ig is IgG
2) IgG is the major Ig in extra vascular spaces
3) Placental transfer - IgG is the only class of Ig that crosses the placenta. Transfer is mediated by
receptor on placental cells for the Fc region of IgG. Not all subclasses cross equally; IgG2 do not
cross well.
4) Fixes complement - Not all subclasses fix equally well; IgG4 does not fix complement
5) Binding to cells - Macrophages, monocytes, PMN's and some lymphocytes have Fc receptors for
Page 33
the Fc region of IgG. Not all subclasses bind equally well; IgG2 and IgG4 do not bind to Fc
receptors. A consequence of binding to the Fc receptors on PMN's, monocytes and macrophages is
that the cell can now internalize the antigen better. The antibody has prepared the antigen for
eating by the phagocytic cells. The term opsonin is used to describe substances that enhance
phagocytosis. IgG is a good opsonin. Binding of IgG to Fc receptors on other types of cells results
in the activation of other functions.
B. IgM
1. Structure - The structure of IgM is presented in Figure 8. IgM normally exists as a pentamer (19S
immunoglobulin) but it can also exist as a monomer. In the pentameric form all heavy chains are
identical and all light chains are identical. Thus, the valence is theoretically 10. IgM has an extra
domain on the chain (CH4) and it has another protein covalently bound via a S-S bond called
the J chain. This chain functions in polymerization of the molecule into a pentamer.
Figure 26: Immunoglobulin J Chain and C4
Properties
a) IgM is the 3rd most common serum Ig.
b) IgM is the first Ig to be made by the fetus and the first Ig to be made by a virgin B cells when
it is stimulated by antigen.
c)
As a consequence of its pentameric structure, IgM is a good complement fixing Ig. Thus, IgM
antibodies are very efficient in leading to the lysis of microorganisms.
d)
As a consequence of its structure, IgM is also a good agglutinating Ig. Thus, IgM antibodies
are very good in clumping microorganisms for eventual elimination from the body.
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e) IgM binds to some cells via Fc receptors.
f)
B cell surface Ig - Surface IgM exists as a monomer and lacks J chain but it has extra 20
amino acids at the C-terminal end to anchor it into the membrane (Figure 27). Cell surface IgM
functions as a receptor for antigen on B cells. Surface IgM is noncovalently associated with two
additional proteins in the membrane of the B cell called Ig- and Ig- as indicated in Figure 10.
These additional proteins act as signal transducing molecules since the cytoplasmic tail of the Ig
molecule itself is too short to transduce a signal. Contact between surface immunoglobulin and an
antigen is required before a signal can be transuded by the Ig- and Ig- chains. In the case of Tindependent antigens, contact between the antigen and surface immunoglobulin is sufficient to
activate B cells to differentiate into antibody secreting plasma cells. However, for T-dependent
antigens, a second signal provided by helper T cells is required before B cells are activated.
Figure 27: Immunoglobulin Tail piece
Figure 28: Immunoglobulin structure and

C IgA
Structure - Serum IgA is a monomer but IgA found in secretions is a dimer as presented in Figure
27. When IgA exits as a dimer, a J chain is associated with it. When IgA is found in secretions is
also has another protein associated with it called the secretory piece or T piece; sIgA is sometimes
referred to as 11S immunoglobulin. Unlike the remainder of the IgA which is made in the plasma
cell, the secretory piece is made in epithelial cells and is added to the IgA as it passes into the
secretions (Figure 28). The secretory piece helps IgA to be transported across mucosa and also
protects it from degradation in the secretions.
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Figure 29: Immunoglobulin secretory piece

Properties
1) IgA is the 2nd most common serum Ig.
2) IgA is the major class of Ig in secretions - tears, saliva, colostrum, mucus. Since it is found in
secretions secretory IgA is important in local (mucosal) immunity.
3) Normally IgA does not fix complement, unless aggregated.
4) IgA can bind to some cells - PMN's and some lymphocytes.
5) IgD
3. Structure - The structure of IgD is presented in the Figure 13. IgD exists only as a monomer.
2. Properties
IgD is found in low levels in serum; its role in serum uncertain.
IgD is primarily found on B cell surfaces where it functions as a receptor for antigen. IgD on the
surface of B cells has extra amino acids at C-terminal end for anchoring to the membrane. It also
associates with the Ig- and Ig- chains.
IgD does not bind complement.
IgE
Structure - The structure of IgE is presented in Figure 31. IgE exists as a monomer and has an
extra domain in the constant region.
Properties
IgE is the least common serum Ig since it binds very tightly to Fc receptors on basophils and mast
cells even before interacting with antigen.
Figure 30: IgE Tail piece

Figure 31: IgE C4
Page 36
Involved in allergic reactions - As a consequence of its binding to basophils an mast cells, IgE
is involved in allergic reactions. Binding of the allergen to the IgE on the cells results in the
release of various pharmacological mediators that result in allergic symptoms. IgE also plays a
role in parasitic helminth diseases. Since serum IgE levels rise in parasitic diseases, measuring
IgE levels is helpful in diagnosing parasitic infections. Eosinophils have Fc receptors for IgE
and binding of eosinophils to IgE-coated helminths results in killing of the parasite.
IgE does not fix complement.
6.. Immunoglobulins: Isotypes, Allotypes and Idiotypes
I. Isotypes
Definition - Isotypes are antigenic determinants that characterize classes and subclasses of heavy
chains and types and subtypes of light chains. If human IgM is injected into a rabbit the rabbit will
recognize antigenic determinants on the heavy chain and light chain and make antibodies to them.
If that antiserum is absorbed with human IgG the antibodies to the light chain determinants and
any determinants in common between human IgM and IgG will be removed and the resulting
antiserum will be react only with human IgM. Indeed, the antibodies will only react with the
constant region of the chain. Antibodies to the variable region are rare perhaps because only a
few copies of each different variable region are represented in the IgM and thus effective
immunization does not occur. The determinants that are recognized by such antibodies are called
isotypic determinants and the antibodies to those determinants are called anti-isotypic antibodies.
Each class, subclass, type and subtype of immunoglobulin has its unique set of isotypic
determinants.
Location - Heavy chain isotypes are found on the Fc portion of the constant region of the molecule
while light chain isotypes are found in the constant region. The location of isotypic determinants is
illustrated in Figure 32.
Figure 32: Ig Isotopes Kappa

Occurrence - Isotypes are found in ALL NORMAL individuals in the species. The prefix Iso
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means same in all members of the species. Some individuals with immunodeficiencies may lack
one or more isotypes but normal individuals have all isotypes.
Importance - Antibodies to isotypes are used for the quantitating Ig classes and subclasses in
various diseases, in the characterization of B cell leukemia and in the diagnosis of various
immunodeficiency diseases.
II.
Allotypes
A. Definition - Allotypes are antigenic determinants specified by allelic forms of the Ig genes.
B. Allotypes represent slight differences in the amino acid sequences in the heavy or light chains of
different individuals.
Even a single amino acid difference can give rise to an allotypic
determinant, although in many cases the several amino acid substitutions have occurred.
C. Allotypic differences are detected by using antibodies directed against allotypic determinants.
These antibodies can be prepared by injecting the Ig from one person into another. In practice
however we obtain anti-allotype antisera from women who have had multiple pregnancies or
from people who have received blood transfusions or from some patients with rheumatoid
arthritis.
B.
Location - In man the allotypic differences are localized to the constant region of the heavy and
light chains as illustrated in the Figure 33.
Figure 33: Ig Allotypes

C.
Occurrence - Individual allotypes are found in individual members of a species. All allotypes are
not found in all members of the species. The prefix Allo means different in individuals of a
species
D.
Human Ig Allotypes
Nomenclature - Human Ig allotypes are named on the basis of the heavy or light chain on which
Page 38
it is located. Thus, an allotype on a Gamma 1 heavy chain is given the name: G1m(3). An
allotype on a Kappa light chain is given the name: Km(1). Table 1 lists some human allotypes.
Genetics
1.
Codominant autosomal genes - Allotypes that represent amino acid substitutions at the same
position in a heavy or light chain (eg. G1m(3) and G1m(17) or Km(1) and Km(3) are inherited as
codominant autosomal genes.
eg.
Km(1)/Km(3)
Km(1)/Km(1)
Km(1)/Km(1) and Km(1)/Km(3)

2.
Allelic Exclusion - Although in a heterozygote both alleles are expressed, any individual Ig
molecule will only have one allotype. This is because an individual B cell can only expresses one
allele. This is called allelic exclusion. Allotypes that represent amino acid substitutions at
different locations in a molecule (eg. G1m(1) and G1m(17)) can be found on the same molecule.
eg. In a G1m(1,17) individual both allotypes can be on the same heavy chain
G1m(17)
Gm1(1)
|
214
F.
|
355-358
Importance
1.
Monitoring bone marrow grafts - Bone marrow grafts that produce a different allotype from the
recipient can be used to monitor the graft.
2.
Forensic medicine - Km and Gm allotypes are detectable in blood stains and semen and are useful
3.
in forensic medicine.
Paternity testing - The immunoglobulin allotypes are one of the characteristics used in legal cases
involving paternity.
III.
A.
Idiotypes (Id)
Definition - Unique antigenic determinants present on individual antibody molecules or on

molecules of identical specificity.
Identical specificity means that all antibodies molecules have the exact same hypervariable
Page 39
regions.
To understand what idiotypes are, it is helpful to understand how they are detected.
DNP-BSA Strain A anti-DNP Ab
?? Strain A purified
anti-DNP Ab
Antigenic determinants created by the combining site of an antibody are called idiotypes and
the antibodies elicited to the idiotypes are called anti-Id antibodies.
Idiotypes are the
antigenic determinants created by the hypervariable regions of an antibody and the antiidiotypic antibodies are those directed against the hypervariable regions of an antibody.
B.
Location - Idiotypes are localized on the Fab fragment of the Ig molecules as illustrated in
Figure 3. Specifically, they are localized at or near the hypervariable regions of the heavy and
light chains. In many instances the actual antigenic determinant (i.e. idiotype) may include some
of the framework residues near the hypervariable region. Idiotypes are usually determinants created
by both heavy and light chain HVR's although sometimes isolated heavy and light chains will
express the idiotype.
C.
Importance
1. V region marker - Id's are a useful marker for a particular variable region.
2.
Regulation of immune responses - there is evidence that immune responses may be regulated
3.
by anti-Id antibodies directed against our own Id's.

Vaccines - In some cases anti-idiotypic antibodies actually stimulate B cells to make antibody and
thus they can be used as a vaccine. This approach is being tried to immunize against highly
dangerous pathogens that cannot be safely used as a vaccine.
4.
Treatment of B cell tumors - Anti-idiotypic antibodies directed against an idiotype on malignant

B cells can be used to kill the cells. Killing occurs because of complement fixation or because
toxic molecules is attached to the antibodies.
Page 40
Figure 34: Ig Idiotypes location

7. Immunoglobulins: Genetics
I. History
Amino acid sequencing data revealed that a single C region could be associated with many
different V regions. Also, it was shown that a single Idiotypes could be associated with different C
regions (eg. IgM and IgG). To explain these data it was suggested that perhaps the two regions of the
Ig molecule were coded for by separate genes and that the V and C region genes were somehow
joined before an Ig molecule was made (i.e. there were two genes for one polypeptide). This was a
revolutionary concept but with the advent of recombinant DNA technology, it has been shown to be
the correct. The Ig heavy and light chains are coded for by three separate gene families each one on
a separate chromosome - one for the heavy chain and one for each of the light chain types. Each
of these gene families has several V region genes and one or more C region genes. The V and C
regions genes are not however immediately adjacent to each other.
II.
Light chain gene families
1.Germ line gene organization - The organization of the and light chain genes in the germ line or
undifferentiated cells is depicted in Figure 1.
Page 41

Figure 35: Light chain gene families

a. Lambda light chains - The gene family is composed of 4 C region genes, one for each subtype
of chain, and approximately 30 V region genes. Each of the V region genes is composed of two
exons, one (L) that codes for a leader region and the other (V) that codes for most of the variable
region. Upstream of each of the C genes there is and additional exon called J (joining). The L, V,
J and C exons are separated by introns (intervening non-coding sequences).
b. Kappa light chains - The light chain gene family contains only one C region gene, since there
is only one type of light chain.
There are many V region genes (approximately 250)
each of which has a leader exon and a V exon. In the gene family there are several J exons
located between the V and C genes. All of the exons are separated by introns.
2.Gene rearrangement and Expression
As a cell differentiates into a mature B cell that will make a light chain, there is
i a rearrangement of the
various genes (exons) and the
th gene begins to be expressed as depicted in Figure 2. As a cell
commits to become a B cell making a light chain, there is a rearrangement of the genes at the DNA
level such that one of the V genes is brought next to one of the J regions. This occurs by a
recombination event which removes the intron between the V and J regions. The selection of
which V gene is used is not totally
tot
random; there is some preference for the use of V genes
nearest to the J regions. However,
However with time all V genes can be used so that all combinations of
V genes and J regions can be generated.
Page 42

Figure 36: Gene rearrangement and Expression
A consequence of this DNA rearrangement is that the gene becomes transcriptionally ac

active because
a promoter (P), which is associated
assoc
with the V gene, is brought close to an enhancer (E), which is
located in the intron between the J and C regions. As transcription initiates from the promoter a
pre-mRNA
mRNA is made which contains sequences from the L, V J and C regions as well as sequences
for the introns between L and V and between J and C (See Figure 36). This pre-mRNA
pre
is processed
(spliced) in the nucleus and the remaining introns are removed. The resulting mRNA has the L, V J
and C exons contiguous.
The mRNA is translated in the cytoplasm and the leader is removed as the protein is transported
into the lumen of the endoplasmic reticulum. The light chain is assembled with a heavy chain in the
endoplasmic reticulum and the Ig is secreted via the normal route of secretory proteins. The region
V region of the mature light chain is coded for by sequences in the V gene and J region and the C
region by sequences in the C gene.
III. Heavy chain gene family
1.Germ line gene organization - The organization of the heavy chain genes is depicted in Figure 3.
In the heavy chain gene family there aree many C genes, one for each class and subclass of Ig.
Each of the C genes is actually composed of several exons, one for each domain and another for
Page 43
the hinge region. In the heavy chain gene family there are many V region genes, each composed
of a leader and V exon. In addition to several J exons, the heavy chain gene family also contains
several additional exons called the D (diversity) exons. All of the exons are separated by introns as
depicted in Figure 37.
Figure 37: Heavy chain gene family

2. Gene rearrangements and expression
As a cell differentiates into a mature B cell that will make a heavy chain, there is a rearrangement
of the various genes segments (exons) and the gene begins to be expressed as depicted in Figures
4 and 5. As a cell commits to become a B cell making a heavy chain, there are two
rearrangements at the DNA level. First, one of the D regions is brought next to one of the J regions
and then one of the V genes is brought next to the rearranged DJ region. This occurs by two
recombination events which remove the introns between the V, D and J regions. As with the light
chains the selection of the heavy chain V gene is not totally random but eventually all of the V
genes can be used.
A consequence of these DNA rearrangements is that the gene becomes transcriptionally active
because a promoter (P), which is associated with the V gene, is brought close to an enhancer (E),
which is located in the intron between the J and C regions. As transcription initiates from the
promoter a pre-mRNA is made which contains sequences from the L, V, D, J C and C regions
as well as sequences for the introns between L and V, between J and C , and between C and C
Page 44
(Figure 38).
Figure 38: VDJ arrangement
Figure 39: VDJ Transcription

Page 45
The pre-mRNA is processed (spliced) in the nucleus and the remaining introns, including
those between the exons in the C genes, are removed See Figure 5). The pre-mRNA can be
processed in two ways, one to bring the VDJ next to the C gene and the other to bring the VDJ
next to the C gene. The resulting mRNAs have the L, V, D, J and C or C
exons
contiguous and will code for a and a chain, respectively.
The mRNAs are translated in the cytoplasm and the leader is removed as the protein is transported
into the lumen of the endoplasmic reticulum. The heavy chain is assembled with a light chain in
the endoplasmic reticulum and the Ig is secreted via the normal route of secretory proteins. The
region V region of the mature heavy chain is coded for by sequences in the V gene, D region and J
region and the C region by sequences in the C gene.
Mechanism of DNA rearrangements
Flanking the V, J and D exons there are unique sequences referred to as recombination signal
sequences (RSS), which
function in recombination. Each
RSS consists of a conserved
nonamer
and
conserved
heptamer that are separated by

either 12 or 23 base pairs as
illustrated in Figure 6. The 12bp
and 23 bp spaces correspond to
one or two turns of the DNA
helix.
Figure 40: Mechanism of DNA rearrangements
Page 46
Recombination only occurs between a 1 turn and a 2 turn signal. In the case of the light chains
there is a 1 turn signal upstream of the J exon and a 2 turn signal downstream of V. In the case of
the light chains there is a 1 turn signal downstream of the V gene and a 2 turn signal
upstream of the J exon.. In the case of the heavy chains there are 1 turn signals on each side of the
D exon and a 2 turn signal downstream of the V gene and a 2 turn signal upstream of the J
exon. Thus, this ensures that the correct recombination events will occur.
The recombination event results in the removal of the introns between V and J in the case of the
light chains or between the V, D, and J in the case of the heavy chains. The recombination
event is catalyzed by two proteins, Rag-1 and Rag-2. Mutations in the genes for these proteins
results in a severe combined immunodeficiency disease (both T and B cells are deficient), since
these proteins and the RSS are involved in generating both the B and T cell receptors for antigen.
Order of gene expression in Ig gene families
An individual B cell only produces one type of light chain and one class of heavy chain. (N.B.
The one exception is that a mature B cell can produce both and heavy chains but the antibody
specificity is the same since the same VDJ region is found on the and chains). Since any B
cell has both maternal and paternal chromosomes which code for the Ig genes there must be some
orderly way in which a cell expresses its Ig genes so as to ensure that only one type of light chain
and one class of heavy chain is produced? The order in which the Ig genes are expressed in a B
cell is depicted in Figure 41.
Figure 41: Ig genes are expressed in a B cell (Heavy chain)

Page 47
Heavy chain (Figure 41) - A cell first attempts to rearrange one of its heavy chain genes; in some
cells the maternal chromosome is selected and in others the paternal chromosome is selected. If the
rearrangement is successful so that a heavy chain is made, then no further rearrangements occur in
the heavy chain genes. If, on the other hand, the first attempt to rearrange the heavy chain genes is
unsuccessful (i.e. no heavy chain is made), then the cell attempts to rearrange the heavy chain genes
on its other chromosome. If the cell is unsuccessful in rearranging the heavy chain genes the second
time, it is destined to be eliminated.
Figure 42: Ig genes are expressed in a B cell (light chain)

Kappa light chain (Figure 42) - When a cell successfully rearranges a heavy chain gene, it then
begins to rearrange one of its light chain genes.
It is a random event whether the
maternal or paternal light chain genes are selected. If the rearrangement is unsuccessful (i.e. it
does not produce a functional light chain), then it attempts to rearrange the genes on the
other chromosome.
If a cell successfully rearranges a light chain gene, it will be a B cell that
makes an Ig with a light chain.

Lambda light chain (Figure 42) - If a cell is unsuccessful in rearranging both of its light chain genes,
it then attempts to make a light chain. It is a random event whether the maternal or paternal
light chain genes are selected. If the rearrangement is unsuccessful (i.e. it does not produce a
functional light chain), then it attempts to rearrange the genes on the other chromosome. If a
cell successfully rearranges a light chain gene, it will be a B cell that makes an Ig with a light
chain.
Page 48
The orderly sequence of rearrangements in the Ig gene families explains:

1)Why an individual B cell can only produce one kind of immunoglobulin with one kind of heavy
and one kind of light chain.
2) Why a individual B cell can only make antibodies of one specificity.
3) Why there is allelic exclusion in Ig allotypes at the level of an individual Ig molecule but codominant expression of allotypes in the organism as a whole.
Origin of Antibody Diversity
Background - Antibody diversity refers to the sum total of all the possible Ab specificities
that an organism can make. It is estimated that we can make 107 - 108 different Ab molecules.
One of the major questions in immunology has been how can we make so many different antibody
molecules. Theories which have attempted to explain the origin of antibody diversity fall into two
major categories.
Germ line theory - This theory states that we different V region gene for each possible antibody
we can make.
Somatic mutation theory - This theory state that we have only one or a few V region genes
and the diversity is generated by somatic mutations which occur in these genes.
Current Concepts - Our current thinking is that both the germ line and somatic mutation
theories have some merit. It is thought that antibody diversity is generated by the following
mechanisms.
1. Large number of V genes
a)
30 lambda V genes b)
300 kappa V genes c)
1000 heavy chain V genes
2.V-J and V-D-J joining - The region where the light chain V gene and J region or the heavy chain V
gene and D and J regions come together is in the 3rd hyper variable region. Since it is random which
V and which J or D regions come together, there is a lot of diversity that can be generated by V-J
and V-D-J joining.
3.Junctional diversity (Inaccuracies in V-J and V-D and D-J recombination) - (Figure 9).
Recombination between V-J and V-D-J is not always perfect and additional diversity can arise
by errors that occur in the recombination event that brings the V region next to the J or D regions
or the D region next to the J region. It is estimated that these inaccuracies can triple the diversity
generated by V-J and V-D-J joining. The diversity generated by this mechanisms is occurring in
the 3rd hypervariable region and thus, is directly affecting the combining site of the Ab.
4.N region insertion - At the junction between D and J segments there is often an insertion
Page 49
of a series of nucleotides which is catalyzed by the enzyme terminal transferase. (Terminal

transferase catalyzes the radon polymerization of nucleotides into DNA without the need for a
template. This leads to further diversity in the 3rd hypervariable region.
5.Somatic Mutation - There is evidence that somatic mutations are occurring in the V gene,
particularly in the place that codes for the 2nd hypervariable region. Thus, somatic mutation
probably contributes to Ab diversity to some extent.
6.Combinatorial Association - Any individual B cell has the potential to make any one of the possible
heavy chains and any one of the possible light chains. Thus, different combinations of heavy and
light chains within an individual B cell adds further diversity.
7.Multispecificity - Due to cross reactions between antigenic determinants of similar structure an
antibody can often react with more than one antigenic determinant. This is termed
multispecificity. Multispecificity also contributes to Ab diversity.
The process of gene rearrangement of the heavy and light chains and the combinatorial association
of these chains occurs during B cell development and is independent of antigen. Clones of B cells
expressing all of the possible antibody specificities are produced during development and antigen
simply selects those clones which have the appropriate receptor.The selected clones are then
activated, proliferate and differentiate into antibody secreting plasma cells.
T Cell Receptor For Antigen
T cells also have a receptor for antigen on their surfaces.
This receptor is not an
immunoglobulin molecule but it is composed of two different polypeptide chains which have
constant and variable regions analogous to the immunoglobulins. Diversity in the T cell receptor
is also generated in the same way as described for antibody diversity (e.g. by VJ and VDJ joining
of gene segments and combinatorial association). However, no somatic mutation has been
observed in T cells.
8. Antibody Formation
8.1. General Characteristics of the Antibody Response
A. Self/non-self discrimination - One characteristic feature of the specific immune system is that
Page 50
it normally distinguishes between self and non-self and only reacts against non-self.
B. Memory - A second feature of the specific immune response is that it demonstrates memory. The
immune system "remembers" if it has seen an antigen before and it reacts to secondary exposures
to an antigen in a manner different than after a primary exposure. Generally only an exposure to the
same antigen will illicit this memory response.
C. Specificity - A third characteristic feature of the specific immune system is that there is a high degree
of specificity in its reactions. A response to a particular antigen is specific for that antigen or a
few closely related antigens.
8.2.. Antibody Formation
A.
Fate of the immunogen
1. Clearance after primary injection - The kinetics of Ag clearance from the body after a primary
administration is depicted in Figure 1.
A. Equilibrium phase - The first phase is called the equilibrium or equilibration phase. During this time
the Ag equilibrates between the vascular and extra vascular compartments by diffusion. This is
normally a rapid process. Since particulate antigens don't diffuse, they do not show this phase.
B. Catabolic decay phase - In this phase the host's cells and enzymes metabolize the antigen. Most
of the antigen is taken up by macrophages and other phagocytic
cells. The duration will
depend upon the immunogen and the host.
Figure 43: antibody formation

Page 51
c) Immune elimination phase - In this phase newly synthesized antibody combines with the
antigen producing antigen/antibody complexes which are phagocytosed and degraded. Antibody
appears in the serum only after the immune elimination phase is over.
2. Clearance after secondary injection - If there is circulating antibody in the serum injection of
the antigen for a second time results in a rapid immune elimination. If the is no circulating antibody
then injection of the antigen for a second time results in all three phases but the onset of the immune
elimination phase is accelerated.
8.3.
.3. Kinetics of antibody responses to T-dependent Ag
1.
Primary (1o) Ab response - The kinetics of a primary antibody response to an antigen is

illustrated in Figure 43.
a) Inductive, latent or lag phase - In this phase the Ag is recognized as foreign and the cells begin to
proliferate and differentiate in response to the antigen. The duration of this phase will vary
depending on the antigen but it is usually 5-7 days.
b) Log or Exponential Phase - In this phase the
Ab concentration
increases exponentially as the
B cells that were stimulated by the antigen differentiate into plasma cells which secrete antibody.
c) Plateau or steady-state phase - In this phase Ab synthesis is balanced by Ab decay so that there in
no net increase in Ab concentration.
d) Decline or decay phase - In this phase the rate of Ab degradation exceeds that of Ab synthesis and
the level of Ab falls. Eventually the level of Ab may reach base line levels.
Page 52
Figure 44: antibody formation after immunization (10 and (20)

2. Secondary (2o), memory or anamnestic response
a).Lag phase - In a secondary response there is a lag phase by it is normally shorter than that
observed in a primary response.
b) Log phase - The log phase in a secondary response is more rapid and higher Ab levels are
achieved.
c) Steady state phase
d)
Decline phase - The decline phase is not as rapid and Ab may persist for months,
years or even a lifetime.
8.4. Specificity of 1o and 2o responses
Ab elicited in response to an antigen is specific for that antigen although it may also cross react
with other antigens which are structurally similar to the eliciting antigen. In general secondary
responses are only elicited by the same antigen used in the primary response. However, in some
instances a closely related antigen may produce a secondary response, but this is a rare
exception.
Page 53
8.5. Qualitative changes in Ab during 1o and 2o responses

1. Ig class variation - In the primary response the major class of Ab produced is IgM whereas in
the secondary response it is IgG (or IgA or IgE) (Figure 4).
The antibodies that
persist
in
the secondary response are the IgG antibodies.

2. Affinity - The affinity of the IgG Ab produced increases progressively during the response,
particularly after low doses of antigen (Figure 5). This is referred to as affinity maturation.
Affinity maturation is most pronounced after secondary challenge with antigen.
Figure 45 : antibody affinity
One explanation for affinity maturation is clonal selection as illustrated in Figure 47. A second
explanation for affinity maturation is that, after a class switch has occurred in the immune response,
somatic mutations occur which fine tunes the antibodies to be of higher affinity. There is
experimental evidence for this mechanism, although it is not known how the somatic mutation
Page 54
mechanism is activated after exposure to antigen.
Figure 46: affinity maturation (1 )
Figure 47: affinity maturation (2 )
Avidity - As a consequence of increased affinity, the avidity of the antibodies increases during the
response.
Cross-reactivity - As a result of the higher affinity later in the response there is also an increase in
detectible cross reactivity. An explanation for why increasing affinity results in an increase in
detectible cross reactivity is illustrated by the following example. If a minimum affinity of 10-6 is
needed to detect a reaction, early in an immune response the reaction of a cross reacting antigen
with an affinity of 10-3 will not be detected. However, late in a response when the affinities
increase 1000 fold, the reaction with both the immunizing and cross reacting antigens will be
detected.
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8.6. Cellular events during 1o and 2o responses to T-dependent Ag

1. Primary response (Figure 49)
a) Lag phase - Clones of T and B cells with
become activated and begin to proliferate.
the
The
appropriate antigen receptors bind antigen,

expanded clones of B cells differentiate into
plasma cells which begin to secrete antibody.

b) Log phase - The plasma cells initially secrete IgM antibody since the C heavy chain gene is
closest to the rearranged VDJ gene. Eventually some B cells switch from making IgM to
IgG, IgA or IgE. As more B cells proliferate and differentiate into antibody secreting cells the
antibody concentration increases exponentially
Figure 48: Cellular events during 1o
Figure 49: Cellular events during 2o responses

c) Stationary phase - As antigen is depleted, T and B cells are no longer activated. In addition,
mechanisms which down regulate the immune response come into play. Furthermore, plasma cells
begin to die. When the rate of antibody synthesis equals t h e r a t e o f a n t i b o d y decay
the stationary phase is reached.
d) Decline phase - When no new antibody is produced because the antigen is no longer present to
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activate T and B cells and the residual antibody slowly is degraded, the decay phase is reached.
2. Secondary response (Figure 50)
Not all of the T and B cells that are stimulated by antigen during primary challenge with antigen
die. Some of them are long lived cells and constitute what is referring to as the memory cell
pool. Both memory T cells and memory B cells are produced and memory T cells survive longer
than memory B cells. Upon secondary challenge with antigen not only are virgin T and B cells
activated, the memory cells are also activated and thus there is a shorter lag time in the secondary
response. Since there is an expanded clone of cells being stimulated the rate of antibody
production is also increased during the log phase of antibody production and higher levels are
achieved. Also, since many if not all of the memory B cells will have switched to IgG (IgA or IgE)
production, IgG is produced earlier in a secondary response. Furthermore since there is an expanded
clone of memory T cells which can help B cells to switch to IgG (IgA or IgE) production, the
predominant class of Ig produced after secondary challenge is IgG (IgA or IgE).
Figure 50: Cellelular events during secondary response
Ab response to T-independent Ag
Responses to T-independent Ag are characterized by the production of almost exclusively IgM Ab
and no secondary response. Secondary exposure to the Ag results in another primary response to the
Ag as illustrated in Figure 52.
G. Class switching
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During an antibody response to a T- dependent antigen a switch occurs in the class of Ig produced
from IgM to some other class (except IgD). Our understanding of the structure of the
immunoglobulin genes helps explain how class switching occurs (Figure 52).
Figure 51.class switching

During class switching another DNA rearrangement occurs between a switch site (S) in the intron
between the rearranged VDJ regions and the C gene and another switch site before one of the
other heavy chain constant region genes. The result of this recombination event is to bring the
VDJ region close to one of the other constant region genes, thereby allowing expression of a new
class of heavy chain. Since the same VDJ gene is brought near to a different C gene and since the
antibody specificity is determined by the hyper variable regions within the V region, the antibody
produced after the switch occurs will have the same specificity as before. Cytokines secreted by T
helper cells can cause the switch to certain isotypes.
Membrane and secreted immunoglobulin
The specificity of membrane immunoglobulin on a B cell and the Ig secreted by the plasma cell
progeny of a B cell is the same. An understanding of how the specificity of membrane and secreted
Ig from an individual B cell can be the same comes from an understanding of immunoglobulin
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genes (Figure 11). There are two potential polyA sites in the immunoglobulin gene. One after the
exon for the last heavy chain domain and the other after the exons that code for the transmembrane domains. If the first polyA site is used, the pre-mRNA is processed t o p r o d u c e a
s e c r e t e d protein. If the second polyA site is used, the pre-mRNA is processed to produce a
membrane form of the immunoglobulin. However, i n a l l cases the same VDJ region is used
and thus the specificity of the antibody remains the same. All C regions genes
have
these
additional membrane p i e c e s a s s o c i a t e d w i t h them and thus after class switching other
classes of immunoglobulins can be s e c r e t e d o r e x p r e s s e d o n t h e surface of B cells.
Figure 52: polyadenylation sites

9. Immuniz
Immunization
Immunization is the means of providing specific protection against most common and damaging
pathogens. The mechanism of immunity depends on the site of the pathogen and also the
mechanism of it pathogenesis. Thus, if the mechanism of pathogenesis involves exotoxin, the
only immune mechanism effective against it would be neutralizing antibodies that would prevent
its binding to the appropriate receptor and promoting its clearance and degradation by phagocytes.
Alternatively, if the pathogen produces disease by other means, the antibody will have to react
with the organism and eliminate by complement-mediated lysis or phagocytosis and intracellular
killing. However, if the organism is localized intracellular, it will not be accessible to antibodies
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while it remains inside and the cell harboring it will have to be destroyed and, only then antibody
can have any effect. Most viral infections and intracellular bacteria and protozoa are examples of
such pathogens. In this case, the harboring cells can be destroyed by elements of cell mediated
immunity or if they cause the infected cell to express unique antigens recognizable by antibody,
antibody-dependent and complement mediated killing can expose the organism to elements of
humoral immunity. Alternatively, cells harboring intracellular pathogen themselves can be
activated to kill the organism. Such is the case with pathogens that have the capability of
surviving within phagocytic cells.
Figure 53: types of immunity

Passive Immunity:
Immunity can be gained, without the immune system being challenged with an antigen, by
transfer of serum or gamma globulins from an immune donor to a non-immune individual.
Alternatively, immune cells from an immunized individual may be used to transfer immunity.
Passive immunity may be acquired naturally or artificially.
Naturally acquired passive immunity: Immunity is transferred from mother to fetus through
placental transfer of IgG or colostral transfer of IgA.
Artificially acquired passive immunity: Immunity is often artificially transferred by injection
with gamma globulin from other individuals or from an immune animal. Passive transfer of
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immunity with immune globulin or gamma globulin is practiced in numerous acute infections
(diphtheria, tetanus, measles, rabies, etc.), poisoning (insect-, reptile-bites, botulism), and as a
prophylactic measure (hypogammaglobulinemia). In these situations, gamma globulin of human
origin is preferable although specific antibodies raised in other species (usually horse) are
effective and used in some cases (e.g., poisoning, diphtheria, tetanus, gas gangrene, botulism,
etc.). While this form of immunization has the advantage of providing immediate protection, it
is effective for a short duration only and often results in pathological complications, such as
serum sickness characterized by rash, fever, arthralgia, vasculitis, nephritis, etc., and
anaphylaxis. Homologous immunoglobulin may carry the risk of transmitting hepatitis and HIV
and other blood borne diseases.
Passive transfer of cell-mediated immunity (immunity that is transferred by cells and not by
antibody) can also be accomplished in certain diseases (cancer, immunodeficiency). However, it
is difficult to find histocompatible (matched) donors and there is severe risk of graft versus host
disease.
Active Immunity:
This refers to immunity produced by the body following exposure to antigens.
Naturally acquired active immunity: Exposure to different pathogens leads to sub clinical or
clinical infections, which normally result in a protective immune response against these
pathogens.
Artificially acquired active immunity: Immunization may be achieved by administering live or
dead pathogens or their components. Vaccines used for active immunization consist of live
(attenuated: capable of producing very mild or no symptoms) organism, killed whole organism,
microbial components or secreted, detoxified toxins (toxoid).
Live vaccines: Live organisms are used for immunization against a number of viral infections.
Live vaccines for measles, mumps, rubella and chicken pox (varicella) are used routinely. A live
bacterial vaccine consisting of a strain of Mycobacterium bovis, Bacillus Calmet Geurin (BCG) is
used against tuberculosis in many African, European and Asian countries but not many others.
Whereas many studies have shown the efficacy of BCG vaccine, a number of studies also cast
doubt on its benefits.
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Live vaccines normally produce self-limiting non-clinical infections and lead to subsequent
immunity, both humoral and cell-mediated, the latter being essential for intracellular pathogens.
However, they carry a serious risk of causing overt disease in immunocompromised individuals.
Furthermore, since live vaccines are often attenuated (made less pathogenic) by passage in animal
or thermal mutation, they can revert to their pathogenic form and cause serious illness. It is for
this reason, polio live (Sabin) vaccine, which was used for many years, has been replaced in many
countries by the inactivated (Salk) vaccine.
Killed vaccines: These consist of whole organisms inactivated by heat, chemicals or UV irradiation
treatment. Many killed viral and bacterial vaccines are available. Some of these are used to
immunize people at risks (e.g. influenza, hepatitis A, etc.) while others are used to immunize
travelers to different countries (e.g. cholera, typhoid etc.). Pertussis (whooping cough) whole
bacterial vaccine was used routinely until a few years ago, but due to its serious side effect, it has
been replaced by a formulation of acellular components.
Sub-unit vaccines: Some vaccines consist of subcomponents of the pathogenic organisms, usually
proteins or polysaccharides. Since polysaccharides are relatively weak T-independent antigens,
and produce only IgM responses without immunologic memory, they are made more
immunogenic and T-dependent by conjugation with proteins (e.g., haemophilus, meningococcus,
pneumococcus, etc.). Hepatitis-B, rabies vaccines consist of antigenic proteins cloned into a
suitable vector (e.g., yeast). These subunit vaccines are designed to reduce the problems of
toxicity and risk of infection. When the pathogenic mechanism of an agent involves a toxin, a
modified form of the toxin (toxoid) is used as vaccine (e.g., diphtheria, tetanus, etc.). Toxoids,
although lose their toxicity, they remains immunogenic.
Other novel vaccines: A number of novel approaches to active immunization are in the
investigative stage and are used only experimentally. These include anti-idiotype antibodies,
DNA vaccines and immunodominant peptides (recognized by the MHC molecules) and may be
available in the future. Anti-idiotype antibodies against polysaccharide antibody produce long
lasting immune responses with immunologic memory. Viral peptide genes cloned into vectors,
when injected transfect host cells and consequently produce a response similar to that produced
against live-attenuated viruses (both cell-mediated and humoral). Immunodominant peptides are
simple and easy to prepare and, when incorporated into MHC polymers, can provoke both
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humoral and cell mediated responses.

Adjuvants: Weaker antigens may be rendered more immunogenic by the addition of other
chemicals. Such chemicals are known as adjuvants. There are many biological and chemical
substances that have been used in experimental conditions (Table 1). However, only Aluminum
salts (alum) are approved for human use and it is incorporated in DTP vaccine. Furthermore,
pertussis itself has adjuvant effects. Adjuvants used experimentally include mixtures of oil and
detergents, with (Freunds complete adjuvant) or without certain bacteria (Freunds incomplete
adjuvant). Bacteria most often used in an adjuvant are Mycobacteria (BCG) and Nocardia. In
some instance sub-cellular fractions of these bacteria can also be used effectively as adjuvants.
Newer adjuvant formulations include synthetic polymers and oligonucleotides. Most adjuvants
recognize TOLL-like receptors thus activating mononuclear phagocytes and inducing selective
cytokines that can enhance Th1 or Th2 responses, depending on the nature of the adjuvant.
Figure 54: Newer adjuvant formulations
The protective immunity conferred by a vaccine may be life-long (measles, mumps, rubella,
small pox, tuberculosis, yellow fever, etc.) or may last as little as a few months (cholera). The
primary immunization may be given at the age of 2-3 months (diphtheria, pertussis, tetanus,
recommended age range polio), or 13-15 months (mumps, measles, rubella). The currently
recommended schedule of routine immunization in the USA (recommended by CDC and AIP)
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is summarized in Figure 55. This schedule is revised on yearly basis or as need by the CDC
Advisory Committee on Immunization Practice (AICP).
Prophylactic versus therapeutic immunization: Most vaccines are given prophylactic ally, i.e.,
prior to exposure to the pathogen. However, some vaccines can be administered therapeutically,
i.e., post exposure (e.g., rabies virus). The effectiveness of this mode of immunization depends on
the rate of replication of the pathogen, incubation period and pathogenic mechanism. For this
reason, only a booster shot with tetanus is sufficient if the exposure to the pathogen is within less
than 10 years and if the exposure is minimal (wounds are relative superficial). In a situation
where pathogen has a short incubation period, the pathogenic mechanism is such that only a small
amount of pathogenic molecules could be fatal (e.g., tetanus and diphtheria) and/or bolus of
infection is relatively large, both passive and active post exposure immunization are essential.
Passive prophylactic immunization is also normal in cases of defects in the immune system, such
as hypogammaglobulinemias.
Adverse effects of immunization: Active immunization may cause fever, malaise and
discomfort. Some vaccine may also cause joint pains or arthritis (rubella), convulsions,
sometimes fatal (pertussis), or neurological disorders (influenza). Allergies to egg may develop
as a consequence of viral vaccines produced in egg (measles, mumps, influenza, yellow fever).
Booster shots result in more pronounced inflammatory effects than the primary immunization.
The noticeable and serious side effects documented have been those following the DTP vaccine
(Table 2). Most of these were attributable to the whole pertussis component of the vaccine and
have been eliminated since the use of the acellular pertussis preparation.
10. Cel
Cells of the Immune System
stem and Antigen Recognition
1) Overview
a)
The immune system has developed to protect the host from pathogens and other foreign
substances. Self/non-self discrimination is one of the hallmarks of the immune system. There are
two mains sites where pathogens may reside: extracellularly in tissue spaces or intracellularly
within a host cell; and the immune system has different ways of dealing with pathogens at these
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sites.
Although immune responses are tailored to the pathogen and to where the pathogen
resides, most pathogens can elicit both an antibody and a cell- mediated response, both of which
may contribute to ridding the host of the pathogen. However, for any particular pathogen an
antibody or a cell-mediated response may be more important for defense against the pathogen
b) Extracellular pathogens: antibodies are the primary defense against extracellular pathogens
and they function in three major ways:
i) Neutralization - by binding to the pathogen or foreign substance antibodies can block the
association of the pathogen with their targets. For example, antibodies to bacterial toxins
can prevent the binding of the toxin to host cells thereby rendering the toxin ineffective.
Similarly, antibody binding to a virus or bacterial pathogen can block the attachment of the
pathogen to its target cell thereby preventing infection or colonization.
ii) Opsonization - Antibody binding to a pathogen or foreign substance can opsonize the material and
facilitate its uptake and destruction by phagocytic cells. The Fc region of the antibody interacts
with Fc receptors on phagocytic cells rendering the pathogen more readily phagocytosed.
iii) Complement activation - Activation of the complement cascade by antibody can result in
lysis of certain bacteria and viruses. In addition, some components of the complement cascade
(e.g. C3b) opsonize pathogens and facilitate their uptake via complement receptors on phagocytic
cells.
c) Intracellular pathogens: Because antibodies do not get into host cells, they are ineffective against
intracellular pathogens. The immune system uses a different approach to deal with these kinds
of pathogens. Cell-mediated responses are the primary defense against intracellular pathogens and
the approach is different depending upon where the pathogen resides in the host cell (i.e., in the
cytosol or within vesicles). For example, most viruses and some bacteria reside in the cytoplasm
of the host cell, however, some bacteria and parasites actually live within endosomes in the
infected host cell.
The primary defense against pathogens in the cytosol is the cytotoxic T
lymphocyte (Tc or CTL). In contrast, the primary defense against a pathogen within vesicles
is a subset of helper T lymphocytes (Th1).
i) Cytotoxic T cells (CTL) - CTLs are a subset of T lymphocytes that express a unique antigen on
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their surface called CD8.
These cells recognize antigens from the pathogen that are
displayed on the surface of the infected cell and kill the cell thereby preventing the spread of the
infection to neighboring cells. CTLs kill by inducing apoptosis in the infected cell.
ii) Th1 helper T cells - Th cells are a subset of T cells that express a unique antigen on their surface
called CD4. A subpopulation of Th cells, Th1 cells, is the primary defense against intracellular
pathogens that live within vesicles. Th1 cells recognize antigen from the pathogen that are
expressed on the surface of infected cells and release cytokines that activate the infected cell.
Once activated, the infected cell can then kill the pathogen. For example, Mycobacterium
tuberculosis, the causative agent of tuberculosis, infects macrophages but is not killed because it
blocks the fusion of lysosomes with the endosomes in which it resides. Th1 cells that recognize
M. tuberculosis antigens on the surface of an infected macrophage can secrete cytokines that
activate macrophages. Once activated the lysosomes fuse with endosomes and the M.
tuberculosis bacteria are killed.
2) Cells of the immune system
a) All cells of the immune system originate from a hematopoietic stem cell in the bone marrow,
which gives rise to two major lineages, a myeloid progenitor cell and a lymphoid
progenitor cell (Figure 1). These two progenitors give rise to the myeloid cells (monocytes,
macrophages, dendritic cells, mast cells, and granulocytes) and lymphoid cells (T cells, B
cells and NK cells), respectively. These cells make up the cellular components of the innate
(non-specific) and adaptive (specific) immune systems.
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Figure 55: stem cell of immune system
b) Cells of the innate immune system Cells of the innate immune system include
phagocytic cells (monocyte/macrophages and PMNs), NK cells, basophils, mast cells, eosinophils
and platelets. The roles of these cells have been discussed previously (see nonspecific immunity,
lecture 1). The receptors of these cells are pattern recognition receptors (PRRs) that recognize
broad molecular patterns found on pathogens (pathogen associated molecular patterns, PAMPS).
c) Cells that link the innate and adaptive immune systems A specialized subset of cells called
antigen presenting cells (APCs) are a heterogeneous population of leukocytes that play an
important role in innate immunity and also act as a link to the adaptive immune system by
participating in the activation of helper T cells (Th cells). These cells include dendritic cells and
macrophages. A characteristic feature of APCs is the expression of a cell surface molecule
encoded by genes in the major histocompatibility complex, referred to as class II MHC molecules.
B lymphocytes also express class II MHC molecules and they also function as APCs, although
they are not considered as part of the innate immune system. In addition, certain other cells
(e.g., thymic epithelial cells) can express class II MHC molecules and can function as APCs.
d) Cells of the adaptive immune system Cells that make up the adaptive (specific) immune system
include the B and T lymphocytes. After exposure to antigen, B cells differentiate into plasma
cells whose primary function is the production of antibodies. Similarly, T cells can differentiate
into either cytotoxic (CTL) or T helper (Th) cells of which there are two types Th1 and Th2
cells. There are a number of cell surface markers that are used in clinical laboratories to
distinguish B cells, T cells and their subpopulations. These are summarized in Table 4.
Table 4: CD Marker cell
Marker
B cell
CTL
Thelper
Antigen R
BCR (surface Ig)
TCR
TCR
CD3
CD4
CD8
CD19/ CD20
CD40
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3) Specificity of the adaptive immune response

a) Specificity of the adaptive immune response resides in the Ag receptors on T and B cells, the
TCR and BCR, respectively. The TCR and BCR are similar in that each receptor is specific for
one antigenic determinant but they differ in that BCRs are divalent while TCRs are monovalent
(Figure 2).
Figure 56: B cell and T cell
b) Each B and T cell has a receptor that is unique for a particular antigenic determinant and there are
a vast array of different antigen receptors on both B and T cells (discussed in more detail in
lecture 11). The question of how these receptors are generated was the major focus of
immunologists for many years. Two basic hypotheses were proposed to explain the generation of
the receptors: the instructionist (template) hypothesis and the clonal selection hypothesis.
i) Instructionist hypothesis The instructionist hypothesis states that there is only one common
receptor encoded in the germline and that different receptors are generated using the Ag as a
template. Each Ag would cause the one common receptor to be folded to fit the Ag. While this
hypothesis was simple and very appealing, it was not consistent with what was known about
protein folding (i.e. protein folding is dictated by the sequence of amino acids in the protein). In
addition this hypothesis did not account for self/non-self discrimination in the immune system. It
could not explain why the one common receptor did not fold around self Ag.
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ii) Clonal selection hypothesis The clonal selection hypothesis states that the germline encodes
many different Ag receptors - one for each antigenic determinant to which an individual will be
capable of mounting an immune response. Ag selects those clones of cells that have the
appropriate receptor. The four basic principles of the clonal selection hypothesis are:
(1) Each lymphocyte has a SINGLE type of Ag receptor with a unique specificity.
(2) Interaction between the foreign molecule and Ag receptor capable of binding that molecule with
a high affinity leads to lymphocyte activation.
(3) The differentiated effector cell derived from an activated lymphocyte will have the same Ag
receptor as the parental lymphocyte; thus they are clones.
(4) Lymphocytes bearing Ag receptors for self molecules are deleted early in lymphoid
development and are absent from the repertoire of mature lymphocytes.
c) The clonal selection hypothesis is now generally accepted as the correct hypothesis to explain how
the adaptive immune system operates. It explains many of the features of the immune
response: 1) the specificity of the response; 2) the signal required for activation of the response
(i.e. Ag); 3) the lag in the adaptive immune response (time is required to activate cells and to
expand the clones of cells); and 4) self/non-self discrimination.
4) Developm
Development of the immune system
a) All immune cells arise from the hematopoietic stem cell. PMNs pass from the circulation into the
tissues. Mast cells are identifiable and thought to be resident in most tissues. B cells mature
in the fetal liver and bone marrow. T cells mature in the thymus. NK cells likely originate
in the bone marrow. Lymphocytes recirculate through secondary lymphoid tissues such as the
spleen where cells such as dendritic cells act as APCs.
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Figure 57: B and T cell origination

5) Lym
Lymphocyte recirculation
a) There are relatively few T or B lymphocytes with a receptor for any particular antigen (1/10,000
1/100,000), the chances for a successful encounter between an antigen and the appropriate
lymphocyte are slim. However, the chances for a successful encounter are greatly enhanced by
the recirculation of lymphocytes through the secondary lymphoid organs.
Lymphocytes
in
the blood enter the lymph nodes and percolate through the lymph nodes (Figure 4). If they
do not encounter an antigen in the lymph node, they leave via the lymphatics and return to the
blood via the thoracic duct. It is estimated that 1-2% of lymphocytes recirculate every hour. If
the lymphocytes in the lymph nodes encounter an antigen, which has been transported to the
lymph node via the lymphatics, the cells become activated, divide and differentiate to become a
plasma cell, Th or CTL cell. After several days the effector cells can leave the lymph nodes via
the lymphatics and return to the blood via the thoracic duct and then make their way to the
infected tissue site.
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Figure 58: lymphatic lymphocytes
b) Nave (virgin) lymphocytes enter the lymph nodes from the blood via High Endothelial Venules
(HEVs). Homing receptors on the lymphocytes direct the cells to the HEVs. In the lymph nodes,
lymphocytes with the appropriate Ag receptor encounter Ag, which has been transported to the
lymph nodes by dendritic cells or macrophages. After activation the lymphocytes express new
receptors that allow the cells to leave the lymph node and reenter the circulation. Receptors on
the activated lymphocytes recognize cell adhesion molecules expressed on endothelial cells near
the site of an infection and chemokines produced at the infection site help attract the activated
cells (Figure 59).
Figure 59: B and T cell differentiation

11. Major Histocompatibility Complex and T Cell Receptors
1) Role of MHC in the im
immune response
a) Cell-cell interactions of the adaptive immune response are critically important in protection
from pathogens.
These interactions are orchestrated by the immunological synapse whose
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primary components are the T cell Ag receptor (TCR) and the Major histocompatibility complex
(MHC) molecule.
The major function of the TCR is to recognize Ag in the correct context of
MHC and to transmit an excitatory signal to the interior of the cell. Since binding of peptide
within the MHC is not covalent, there are many factors while help stabilize the immunological
synapse.
b) There are two types of MHC (class I and class II) which are recognized by different subsets of T
cells. The cytotoxic T cell (CTL) recognizes Ag peptide in the context of MHC class I. The T
helper cell (Th) recognizes Ag presented in MHC class II.
2) Structu
Structure of MHC class I
a) The molecule: Class I MHC molecules are composed of two polypeptide chains, a long chain
and a short chain called 2-microglobulin (60). The chain has four regions. First, a
cytoplasmic region, containing sites for phosphorylation and binding to cytoskeletal elements.
Second, a transmembrane region containing hydrophobic amino acids by which the molecule is
anchored in the cell membrane. Third, a highly conserved 3 immunoglobulin (Ig)-like domain to
which CD8 binds. Fourth, a highly polymorphic peptide binding region formed from the 1 and
2 domains. The 2- microglobulin associates with the chain and helps maintain the proper
conformation of the molecule.
A
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Figure 60: Structure of MHC class I
b) The Ag-binding groove: An analysis of which part of the class I MHC molecules is most variable
demonstrates that variability is most pronounced in the 1 and 2 domains, which comprise
the peptide binding region (Figure 61B). The structure of the peptide binding groove, revealed by
X-ray crystallography, shows that the groove is composed of two helices forming a wall on each
side and eight -pleated sheets forming a floor. The peptide is bound in the groove and the
residues that line the groove make contact with the peptide. These are the residues that are
the most polymorphic. The groove will accommodate peptides of approximately 8-10 amino
acids long. Whether a particular peptide will bind to the groove will depend on the amino acids
that line the groove. Because class I molecules are polymorphic, different class I molecules will
bind many different peptides. Each class I molecule will bind only certain peptides and will have
a set of criteria that a peptide must have in order to bind to the groove. For every class I
molecule, there are certain amino acids that must be a particular location in the peptide before it
will bind to the MHC molecule. Interactions at the N and C-terminus of the peptide are critical
and lock the peptide within the grove. These sites in the peptide are referred to as the anchor
sites. The ends of the peptide are buried within the closed ends of the class I binding
groove while the center bulges out for presentation to the TCR.
3)) Structu
Structure of MHC class II
a) The molecule: Class II MHC molecules are composed of two polypeptide chains, an and a
chain of approximately equal length (Figure 62). Both chains have four regions: first, a
cytoplasmic region containing sites for phosphorylation and binding to cytoskeletal
elements; second, a transmembrane region containing hydrophobic amino acids by which the
molecule is anchored in the cell membrane, third, a highly conserved 2 domain and a highly
conserved 2 domain to which CD4 binds and fourth, a highly polymorphic peptide binding
region formed from the 1 and 1 domains.
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Figure 61: Structure of MHC class II

b) The Ag-binding groove: As with Class I MHC molecules, an analysis of which part of the class II
MHC molecule is most variable demonstrates that variability is most pronounced in the 1 and 1
domains, which comprise the peptide binding region (Figure 62 A). The structure of the peptide
binding groove, revealed by X-ray crystallography, shows that, like class I MHC molecules, the
groove is composed of two helices forming a wall on each side and eight -pleated sheets
forming a floor. Both the 1 and 1 chain contribute to the peptide binding groove. The peptide
is bound in the groove and the residues that line the groove make contact with the peptide. These
are the residues that are the most polymorphic. The groove of Class II molecules is open at one
end so that the groove can accommodate longer peptides of approximately 13-25 amino acids long
with some of the amino acids located outside of the groove. Whether a particular peptide will
bind to the groove will depend on the amino acids that line the groove. Because class II
molecules are polymorphic, different class II molecules will bind different peptides. Like
class I molecules, each class II molecule will bind only certain peptides and will have a set of
criteria that a peptide must have in order to bind to the groove (i.e. anchor sites).
4) Important aspects of MHC
a) Although there is a high degree of polymorphism for a species, an individual has maximum of six
different class I MHC products and only slightly more class II MHC products (considering only
the major loci). Each MHC molecule has only one binding site. The different peptides a given
MHC molecule can bind all bind to the same site, but only one at a time. Because each MHC
molecule can bind many different peptides, binding is termed degenerate. MHC polymorphism is
determined only in the germline. There are no recombinatorial mechanisms for generating
diversity. MHC molecules are membrane-bound; recognition by T cells requires cell-cell contact.
Alleles for MHC genes are co-dominant. Each MHC gene product is expressed on the cell
surface of an individual nucleated cell. A peptide must associate with a given MHC of that
particular individual otherwise no immune response can occur. Mature T cells must have a T cell
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receptor that recognizes the peptide associated with MHC. Cytokines (especially interferon-)
increase level of expression of MHC. Polymorphism in MHC is important for survival of the
species.
b) How do peptides get into the MHC groove (Figure 62)? Peptides from the cytosol associate
with class I MHC and are recognized by CTL cells. The peptides enter the endoplasmic
reticulum and bind in the MHC class I groove. This complex is then exported to the cell
surface through the golgi. MHC class II molecules are formed with an invariant (Ii) chain as a
place holder while in the ER and Golgi. The Ii chain is cleaved and removed once the complex
is in a vesicle. Peptides from within the vesicle associate with class II MHC and are then
exported to the cell surface where they are recognized by Th cells.
5) Role of TCR in the immune response
a) The TCR is a surface molecule found on T cells that recognizes Ag presented in the correct
MHC context. The TCR is similar to immunoglobulin (Ig) and is part of the Ig superfamily.
There are two types of TCRs, the predominant which is commonly found in lymphoid
tissues, and the which is found at mucosal surfaces.
6) Structure of the TCR ()
Figure 62: Role of TCR in the immune response

The TCR is a heterodimer composed of one and one chain of approximately equal length
(Figure 64). Each chain has a short cytoplasmic tail but it is too small to be able to transduce an
activation signal to the cell. Both chains have a transmembrane region comprised of hydrophobic
amino acids by which the molecule is anchored in the cell membrane.
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Figure 63: TCR a heterodimer

Both chains have a constant region and a variable region similar to the immunoglobulin chains.
The variable region of both chains contains hyper variable regions that determine the specificity
for antigen.
6) Important aspects of the TCR
a) Each T cell bears a TCR of only one specificity (i.e. there is allelic exclusion). The TCR
recognizes Ag only in the context of cell-cell interaction and in the correct MHC. The TCR
recognizes Ag in an MHC-independent manner in response to certain viral and bacterial Ag.
8)) Genetic basis for recep
receptor generation
a) The genetic basis for the generation of the vast array of antigen receptors on B cells has been
discussed previously (see lecture on Ig genetics). The generation of a vast array of TCRs is
accomplished by similar mechanism. The germline genes for the TCR genes are composed of
V, D and J gene segments that rearrange during T cell development to produce many different
TCR chains (Figure 64). The germline genes for the TCR gene are composed of V and
J gene segments which rearrange to produce chains. The specificity of the TCR is determined
by the combination of and chains.
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Figure 64: TCR chains

9) TCR and CD3 complex
a) The TCR is closely associated with a group of 5 proteins collectively called the CD3 complex
(Figure 65). The CD3 complex is composed of one , one , two and 2 chains.
All of the proteins of the CD3 complex are invariant and they do not contribute to the Ag
specificity in any way. The CD3 complex is necessary for cell surface expression of the TCR
during T cell development as it stabilizes the receptor. In addition, the CD3 complex
transduces activation signals to the cell following antigen interaction with the TCR.
Figure 65: Antigen presenting cell
Figure 66: TCR and CD3 Recognition
10) The imm

immunological synapse
synapse
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a) The interaction between the TCR and MHC molecules are not very strong. Accessory molecules
are necessary to help stabilize the interaction (Figure 66 and 67).
These include: 1) CD4
binding to Class II MCH, which ensures that Th cells only interact with APCs; 2) CD8 binding to
Class I MHC, which ensures that CTL cells can interact with target cells; 3) CD2 binding to
LFA-3; and 4) LFA-1 binding to ICAM-1. The accessory molecules are invariant and do not
contribute to the specificity of the interaction, which is solely determined by the TCR. The
expression of accessory molecules can be increased in response to cytokine, which is one way
that cytokines can modulate immune responses.
b) In addition to accessory molecules which help stabilize the interaction between the TCR and
antigen in association with MHC molecules, other molecules are also needed for T cell
activation. Two signals are required for T cell activation one is the engagement of the TCR
with Ag/MHC and the other signal comes from the engagement of co- stimulatory molecules with
their ligands. One of the most important (but not the only) co-stimulatory molecule is CD28
on T cells which must interact with B7-1 (CD80) or B7-2 (CD86) on APCs. Like accessory
molecules the co-stimulatory molecules are invariant and do not contribute to the specificity of the
interaction.
The multiple interactions of TCR with Ag/MHC and the accessory and costimulatory molecules with their ligands have been termed the immunological synapse.
c) Not only is co-stimulation necessary for T cell activation, a lack of co-stimulation may result in
energy (i.e., inability to respond to antigen) or down-regulation of the response. There are a
number of possible outcomes of a T cell receiving one or both of the signals necessary for
activation. Engagement of the TCR with Ag/MHC but no co-simulation results in energy.
Engagement of only the co-stimulatory molecule has no effect. Engagement of TCR with
Ag/MHC and co-stimulatory molecules with their ligand results in activation. Engagement
of the TCR with Ag/MHC and engagement of B7 ligand with CTLA-4, molecules similar to
CD28, results in down-regulation of the response. CTLA-4/B7 interaction sends an inhibitory
signal to the T cell rather than an activating signal. This is one of the ways that immune
responses are regulated. CTLA-4 is expressed on T cells later in an immune response and
this helps to turn off the response.
11) Key steps in T cell activ
ctivation
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a) The APC must process and present peptides to T cells. T cells must receive a co- stimulatory
signal, usually from CD28/CD80 or CD86 interaction. Accessory adhesion molecules must help
to stabilize the binding of T cells and the APC. Signals from the cell surface must be
transmitted to the nucleus via second messengers. Cytokines, produced by the activated cell, help
to drive cell proliferation.
12. Antigen Process and Present

entation
1) Comparison of BCR and TCR
a) B cells and T cells recognize different substances as antigens and in a different form. The B cell
uses cell surface-bound immunoglobulin as a receptor and the specificity of that receptor is the
same as the immunoglobulin that it is able to secrete after activation. B cells recognize the
following antigens in soluble form: 1) proteins (both conformational determinants and
determinants exposed by denaturation or proteolysis); 2) nucleic acids; 3) polysaccharides; 4)
some lipids; 5) small chemicals (haptens).
b) In contrast, the overwhelming majority of antigens for T cells are proteins, and these must be
fragmented and recognized in association with MHC products expressed on the surface of
nucleated cells, not in soluble form. T cells are grouped functionally according to the class of
MHC molecules that associate with the peptide fragments of protein: helper T cells recognize
only those peptides associated with class II MHC molecules, and cytotoxic T cells recognize only
those peptides associated with class I MHC molecules.
2) Ag processing and presentation
a)
Antigen processing and presentation are processes that occur within a cell that result in
fragmentation (proteolysis) of proteins, association of the fragments with MHC molecules,
and expression of the peptide-MHC molecules at the cell surface where they can be recognized by
the T cell receptor on a T cell. However, the path leading to the association of protein fragments
with MHC molecules differs for class I and class II MHC. MHC class I molecules present
degradation products derived from intracellular (endogenous) proteins in the cytosol. MHC class
Page 79
II molecules present fragments derived from extracellular (exogenous) proteins that are located in
an intracellular compartment.
b) MHC class I pathway - All nucleated cells express class I MHC. As shown in Figure 1, proteins
are fragmented in the cytosol by proteosomes (a complex of proteins having proteolytic activity)
or by other proteases. The fragments are then transported across the membrane of the endoplasmic
reticulum by transporter proteins. (The transporter proteins and some components of the
proteosome have their genes in the MHC complex). Synthesis and assembly of class I
heavy chain and beta2 microglobulin occurs in the endoplasmic reticulum. Within the
endoplasmic reticulum, the MHC class I heavy chain, beta2microglobulin and peptide form a
stable complex that is transported to the cell surface.
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Figure 67: MHC class I pathway
c) MHC class II pathway - Whereas all nucleated cells express class I MHC, only a limited group of
cells express class II MHC, which includes the antigen presenting cells (APC). The principal APC
are macrophages, dendritic cells (Langerhans cells), and B cells, and the expression of class II
MHC molecules is either constitutive or inducible, especially by interferon-gamma in the case of
macrophages. As shown in Figure 69, exogenous proteins taken in by endocytosis are fragmented
by proteases in an endosome. The alpha and beta chains of MHC class II, along with an invariant
chain, are synthesized, assembled in the endoplasmic reticulum, and transported through the Golgi
and trans-Golgi apparatus to reach the endosome, where the invariant chain is digested, and
the peptide fragments from the exogenous protein are able to associate with the class II MHC
molecules, which are finally transported to the cell surface.
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Figure 68: MHC class II pathway
d) Important aspects of Ag processing - One way of rationalizing the development of two different
pathways is that each ultimately stimulates the population of T cells that is most effective in
eliminating that type of antigen. Viruses replicate within nucleated cells in the cytosol and
produce endogenous antigens that can associate with MHC class I. By killing these infected cells,
CTL cells help to control the spread of the virus. Bacteria mainly reside and replicate
extracellularly. By being taken up and fragmented inside cells as exogenous antigens that can
associate with MHC class II molecules, helper Th2 T cells can be activated to assist B cells to
make antibody against bacteria, which limits the growth of these organisms. Some bacteria grow
intracellularly inside the vesicles of cells like macrophages. Inflammatory Th1 T cells help to
activate macrophages to kill the intracellular bacteria. Fragments of self, as well as nonself, proteins associate with MHC molecules of both classes and are expressed at the cell
surface. Which protein fragments bind is a function of the chemical nature of the groove for that
specific MHC molecule.
3) Self MHC restriction
a)
In order for a T cell to recognize and respond to a foreign protein antigen, it must
recognize the MHC on the presenting cell as self MHC. This is termed self MHC restriction.
Helper T cells recognize antigen in context of class II self MHC. CTL cells recognize antigen in
context of class I self MHC. The process whereby T cells become restricted to recognizing self
MHC molecules occurs in the thymus.
4) Ag presenting cells (APCs)
a) The three main types of antigen presenting cells are dendritic cells, macrophages and B cells,
although other cells, that express class II MHC molecules, (e.g., thymic epithelial cells) can act
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as antigen presenting cells in some cases. Dendritic cells, which are found in skin and other
tissues, ingest antigens by pinocytosis and transport antigens to the lymph nodes and spleen. In
the lymph nodes and spleen they are found predominantly in the T cells areas. Dendritic cells are
the most effective antigen presenting cells and can present antigens to nave (virgin) T cells.
Furthermore, they can present internalized antigens in association with either class I or class II
MHC molecules (cross presentation), although the predominant pathway for internalized antigen is
the class II pathway. The second type of antigen presenting cell is the macrophage. These cells
ingest antigen by phagocytosis of pinocytosis. Macrophages are not as effective in presenting
antigen to nave T cells but they are very good in activating memory T cells. The third type of
antigen presenting cell is the B cell. These cells bind antigen via their surface Ig and ingest
antigens by pinocytosis. Like macrophages these cells are not as effective as dendrite cells in
presenting antigen to nave T cells. B cells are very effective in presenting antigen to
memory T cells, especially when the antigen concentration is low because surface Ig on the B
cells binds antigen with a high affinity.
5) Presentation of superAg
a) Superantigens are antigens that can polyclonally activate T cells (see lecture on antigens) to
produce large quantities of cytokines that can have pathological effects. These antigens
must be presented to T cells in association with class II MHC molecules but the antigen does not
need to be processed. In the case of a superantigen the intact protein binds to class II MHC
molecules and to one or more V regions of the TCR. The antigen is not bound to the
peptide binding groove of the MHC molecule or to the antigen binding site of the TCR.
Thus, any T cell that uses a particular V in its TCR will be activated by a superantigen,
resulting in the activation of a large numbers of T cells. Each superantigen will bind to a
different set of V regions.
6) Thym
Thymic education
a) Both Th and CTL cells are self-MHC restricted. In addition, T cells do not normally
recognize self antigens. How are self MHC restricted T cells generated and why are self reacting
T cells not produced?
Random VDJ rearrangements in T cells would be expected to
generate some T cells that can recognize non-self MHC and some T cells that can recognize self
Page 83

antigens. It is the role of the thymus to ensure that the only T cells that get to the periphery are
self-MHC restricted and unable to react with self antigen.
Functional T cells in the periphery
periph
have to recognize foreign antigens associ
ssociated with self MHC,
because APC or target cells prese
present foreign antigen associated with self MHC. However, an
individual does not need functional
function T cells in the periphery that recognize
nize antigen (self or foreign)
associated with foreign MHC. An individual especially does not want functional T cells in the
periphery that can recognize self antigens
ens associated with self MHC because they could lead to
damage of healthy, normal
al tissues.
b) As a result of random VDJ recombination
reco
events occurring
ing in immature T cells within the thymus,
thy
TCRs of all specificities are produced. Processes in the thymus determine
deter
which TCR
specificities are retained. There are two sequential steps shown in Figure 3. First, T cells with
the ability to bind to self MHC
M
molecules expressed by cortical thyymic epithelial cells are
retained. This is known as positive
positi selection. Those that do not bind, undergo apoptosis. Thus, T
cells having a TCR that recognizes self MHC survive. Next, T cells with the ability to bind to
self MHC molecules associated
ociated with self molecules expressed by thymic epithelial cells, dendritic
cells and macrophages are killed. This is known as negative selection. Those that do not bind
are retained. As a result of these two steps, T cells having a TCR that recognizes
recogn
self MHC and
foreign antigen survive. Each T cell that survives positive and negative selection in the thymus
and is released into thee periphery retai
retains its specific T cell receptor (TCR).
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Figure 69: random VDJ recombination

reco
c) While positive and negative selection is occurring in the thymus the imm
mature T cells are also
expressing CD4 or CD8 antigens
antige on their surface. Initially the pre-T cell that enters the thymus
is CD4-CD8-. In the thym
mus it becomes CD4+CD8+ and as positive and negative selection
precedes a cell becomes eithher a CD4+ or CD8+ cell. The commitment to become
beco either a CD4+
or CD8+ cells depends on which class of MHC molecule the cell encounters. If a CD4+CD8+
cell is presented
ted with a class I molecule it will down regulate CD4 and become
beco a CD8+ cell. If
a cell is presented with a class II MHC molecule it will down regulate
gulate CD8 and become a CD4+
cell.
Page 85
Figure 70: TCP selection

7) Negative selection in the periphery
a) Positive and negative selection in the thymus is not a 100% efficient process. In addition, not all
self antigens may be expressed in the thymus. Thus some self reactive T cells may get to the
periphery. Thus, there are additional mechanisms that are designed to eliminate self reactive T
cells in the periphery. These will be discussed in the tolerance lecture.
8)) B cell se
select
lection
ction
a) Since B cells are not MHC-restricted there is no need for positive selection of B cells.
However, negative selection (i.e., elimination of self-reactive clones) of B cells is required.
This occurs during B cell development in the bone marrow. However, negative selection of B
cells is not a critical as for T cells since, in most instances, B cells require T cell help in order to
become activated. Thus, if a self reactive B cell does get to the periphery it will not be activated
due to lack of T cell help
13. Cel
Cell-Cel
Cell Inte
Interactions in Immune Res
Responses
1) Central role of Th cells in immune response
a) As depicted in Figure 1, after Th cells recognize specific antigen presented by an APC, they can
initiate several key immune processes. These include: 1) selection of appropriate effector
mechanisms (e.g., B cell activation or CTL generation); 2) induction of proliferation of
appropriate effector cells and 3) enhancement of the functional activities of other cells (e.g.,
Page 86
granulocytes, macrophages, NK cells).
Figure 71: Central role of Th cells in immune response
b) There are four subpopulations of Th cells, Th0, Th1, Th2, and Th17 cells. When nave Th0
cells encounters Ag in secondary lymphoid tissues, they are capable of differentiating
into inflammatory Th1 cells, helper Th2 cells, or pathogenic Th17 cells, which are distinguished by
the cytokines they produce (Figure 73). Whether a Th0 cells becomes a Th1, Th2, or Th17 cell
depends upon the cytokines in the environment, which is influenced by Ag. For example some
antigens stimulate IL-4 production which favors the generation of Th2 cells while other antigens
stimulate IL-12 production, which favors the generation of Th1 cells. Th1, Th2, and Th17 cells
affect different cells and influence the type of immune response, as shown in Figure 3 for Th1 and
Th2.
i) Cytokines produced by Th1 cells activate macrophages and participate in the generation
of CTL cells, resulting in a cell-mediated immune response.
ii) In contrast cytokines produced by Th2 cells help to activate B cells, resulting in antibody
production. In addition, Th2 cytokines also activate granulocytes.
iii) A relatively recent discovery, Th17 cells (designated as such by their production of IL-17)
differentiate (in humans) in response to IL-1, IL-6, and IL-23 (TGF- is important for Th17
differentiation in mice, although not in humans). IL-17 enhances the severity of some
autoimmune diseases including MS, inflammatory bowel disease, and rheumatoid arthritis.
Page 87

Figure 72:: Cytokine production
d) Equally important, each subpopulation can exert inhibitory influences on the other. IFN-
produced by Th1 cells inhibits proliferation of Th2 and differentiation of Th17 cells and IL-10
produced by Th2 cells inhibits production of IFN- by Th1 cells. In addition, although not
shown, IL-4 inhibits production of Th1 and differentiation of Th17 cells. Thus, the immune
response is directed to the type of response that is required to deal with the pathogen encountered
cell-mediated responses
sponses for intracellular
intrac llular pathogens or antibody responses for extracellular
pathogens.
Page 88

2) Cell-cell interactions in Ab responses to exogenous T-dependent

T
Ag a)
Hapten-carrier model:
i) Historically one of the major
ajor findings in immunology was that both T cells and B cells were
required for antibody production to a complex protein.
A major
ajor contribution to our
understanding of this process ca
came from studies on the formation of anti-hapten antibodies.
Studies with hapten-carrier
carrier conjugates established that: 1) Th2 cells recognized the carrier
determinants
ants and B cells recognized haptenic deter
determinants; 2) interactions
tions between haptenspecific B cells and carrier-specific
specific Th cells was self MHC restricted; and 3) B cells can function
both in antigen recognition and in antigen presentation.
ii) B cells occupy a unique position
positi in immune responses because they express immunoglobulin (Ig)
and class II MHC molecules
cules on their
t
cell surface. They therefore are capable of producing
antibody having the samee specificity as that expressed by their immunoglobulin receptor;
recept
in
addition they can function as an antigen presenting cell.
In terms of the hapten-carrier
conjugate model, the mechani
echanism is thought to be the following: the hapten is recognized by the
Ig receptor, the hapten-carrier
carrier is brought into the B cell, processed, and peptide fragments of the
carrier protein are presented
ented to a helper T cell (Figure 4A). Activation of the T cell results in the
production of cytokines that enable the hapten-specific B cell to become activated
acti
to produce
soluble anti-hapten antibodies
ies (Figure
(Fig 74 A).
A.
Page 89
B.
Figure 73: B cell proliferation
iii) Note that there are multiple signals delivered to the B cells in this model of Th2 cell- B cell
interaction. As was the case for activation of T cells where the signal derived from the TCR
recognition of a peptide-MHC molecule was by itself insufficient for T cell activation, so too for
the B cell. Binding of an antigen to the immunoglobulin receptor delivers one signal to the B
cell, but that is insufficient. Second signals delivered by co-stimulatory molecules are required;
the most important of these is CD40L on the T cell that binds to CD40 on the B cell to initiate
delivery of a second signal.
b) Cell-cell interactions in primary Ab response
i) B cells are not the best antigen presenting cell in a primary antibody response; dendritic
cells or macrophages are more efficient. Nevertheless, with some minor modifications the haptencarrier model of cell-cell interactions described above also applies to interactions in a primary
antibody response (Figure 75). In a primary response the Th2 cell first encounters antigen
presented by dendritic cells or macrophages. The primed Th2 cell can then interact with B
cells that have encountered antigen and are presenting antigenic peptides in association with class
II MHC molecules. The B cells still requires two signals for activation one signal is the
binding of antigen to the surface Ig and the second signal comes from CD40/CD40 ligand
engagement during Th2/B cell-cell interaction. In addition, cytokines produced by the Th2 cells
help B cells proliferate and differentiate into antibody secreting plasma cells.
Page 90

Figure 74: Cell-cell

cell interactions in primary Ab response
c) Cell-cell
cell interactions in secondary Ab response
i)
As a consequence of a primary response, many memory T and B cells are produced.
Memory
ory B cells have a high affinity Ig receptor
r
(due to affinity maturation),
tion), which allows them to
bind and present antigen at much lower concentrations than that required for macrophages or
dendritic cells. In addition, memory T cells are more easily activated
ated than nave T cells.
Thus, B/Th cell interactions are sufficient to generate secondary antibody responses. It is not
necessary (although it can occur) to prime memory Th cells with
ith antigen presented by
dendritic cells or macrophages.
acrophages.
ii) Cytokines and class switching: cytokines produced by activated Th2 cells not only stimulate
proliferation and differentiation of B cells, they also help regulate the class of Ab produced.
Different cytokines influence the switch to different classes of Ab with different effort functions
(Table 5). In this way the antibody response is tailored to suit the pathogen encountered (e.g. IgE
antibodies for parasitic worm infections).
Table 5: types of cytokines
Cytokine
IgG1
IgG2a
IL-4
IgG2b
IgG3
IL-5
IFN-
IgA
IgE
IgM
TGF-
Page 91
3) Cell-cell interactions in Ab responses to exogenous T-independent Ag

a)
Antibody responses to T-independent antigens do NOT require cell-cell interactions. The

polymeric nature of these antigens allows for cross-linking of antigen receptors on B cells
resulting in activation. No secondary responses, affinity maturation or class switching occurs.
Responses to T-independent antigens are due to the activation of a subpopulation of B cells
called CD5+ B cells (also called B1 cells), which distinguishes them from conventional B cells
that are CD5- (also called B2 cells).
b) CD5+ cells are the first B cells to appear in ontogeny. They express surface IgM but little
or no IgD and they produce primarily IgM antibodies from minimally somatically mutated germ
line genes. Antibodies produced by these cells are of low affinity and are often polyreactive
(bind multiple antigens). Most of the IgM in serum is derived from CD5+ B cells. CD5+ B
cells do not give rise to memory cells. An important characteristic of these cells is that
they are self-renewing, unlike conventional B cells which must be replaced from the bone
marrow. CD5+ B cells are found in peripheral tissues and are the predominant B cell in the
peritoneal cavity. B1 cells are a major defense against many bacterial pathogens that
characteristically have polysaccharides in their cell walls. The importance of these cells in
immunity is illustrated by the fact that many individuals with T cell defects are still able to resist
many bacterial pathogens
4) Cell-cell interactions in cell-mediated immune response: generation of CTL in response to
exogenous Ag in cytosol
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Figure 75: Process of Antigen presentation
a) Cytotoxic T lymphocytes are not fully mature when they exit the thymus. They have a functional
TCR that recognizes antigen, but they cannot lyse a target cell. They must differentiate into fully
functional effector CTL cells. Cytotoxic cells differentiate from a "pre-CTL" in response to two
signals: specific Ag in the context of MHC class I on a stimulator cell, and cytokines produced
by Th1 cells (especially IL-2 and IFN-) (Fig 75).
b) Features of CTL-mediated lysis- CTL killing is Ag-specific. To be killed by a CTL, the target
cell must bear the same class I MHC-associated Ag that triggered pre-CTL differentiation. CTL
killing requires cell contact. CTL are triggered to kill when they recognize the target Ag
associated with a cell surface MHC molecule. Adjacent cells lacking the appropriate target
MHC-Ag are not affected. CTLs are not injured when they lyse target cells; therefore, each CTL
is capable of killing sequentially numerous target cells.
c)
Mechanisms of CTL killing - CTLs utilize several mechanisms to kill target cells, some of
which require direct cell-cell contact and others that result from the production of certain
cytokines. In all cases death of the target cells is a result of apoptosis.
i) Fas- and TNF-mediated killing (Figure 76): Once generated CTLs express Fas ligand on their
surface, which binds to Fas receptors on target cells. In addition, TNF- secreted by CTLs can
bind to TNF receptors on target cells. The Fas and TNF receptors are a closely related family of
receptors, which when they encounter their ligands, form trimers of the receptors. These receptors
also contain death domains in the cytoplasmic portion of the receptor, which
trimerization can activate caspases that induce apoptosis in the target cell.
after
Page 93
Figure 76: Fas- and TNF-mediated killing

ii) Granule-mediated killing (Figure 77): Fully differentiated CTLs have numerous granules
that contain perforin and granzymes. Upon contact with target cells, perforin is released
and it polymerizes to form channels in the target cell membrane. Granzymes, which are serine
proteases, enter the target cell through the channels and activate caspases and nucleases in the
target cell resulting in apoptosis.
Figure 77: Granule-mediated killing
Page 94
5) Cell-cell interactions in cell-mediated immune response: activation of macrophages in

response to endogenous Ag in vesicles
a) Macrophages play a central role in the immune system. They are involved in 1) the initial
defense against pathogens as part of the innate immune system, 2) Ag presentation to Th cells,
and 3) various effector functions (e.g., cytokine production, bactericidal and tumoricidal
activities) (Figure 78). Indeed macrophages play an important role not only in immunity but also
in reorganization of tissues. However, because of their potent activities, macrophage can also
do damage to tissues.
Figure 78: cytokine production, bactericidal and tumoricidal activities

b)
Many of these macrophage functions can only be performed by activated macrophages.
Macrophage activation can be defined as quantitative alterations in the expression of various gene
products that enable the activated macrophage to perform some function that cannot be
performed by the resting macrophage.
Page 95
c) Macrophage activation is an important function of Th1 cells. When Th1 cells get activated by
an APC such as a macrophage, they release IFN-, which is one of two signals required to
activate a macrophage. Lipopolysaccharide (LPS) from bacteria or TNF- produced by
macrophages exposed to bacterial products deliver the second signal.
d) As discussed in the lecture on the innate immune response (lecture 1), effector
mechanisms employed by macrophages include production of 1) TNF-, which can
induce apoptosis, 2) nitric oxide and other reactive nitrogen intermediates, 3) reactive oxygen
intermediates, and 4) cationic proteins and hydrolytic enzymes.
e) Macrophage activation by Th1 cells is very important in protection against many different
pathogens. For example, Pneumocystis carinii, an extracellular pathogen, is controlled in
normal individuals by activated macrophages; it is, however, a common cause of death in AIDS
patients because they are deficient in Th1 cells. Similarly, Mycobacterium tuberculosis, an
intracellular pathogen that resides in vesicles, is not efficiently killed by macrophages unless
they are activated; hence this infection is a problem in AIDS patients.
14. Cytokines
1) Cytokines are a diverse group of non-antibody proteins that act as mediators between cells.
They were initially identified as products of immune cells that act as mediators and
regulators of immune processes but many cytokines are now known to be produced by cells other
than immune cells and they can have effects on non-immune cells as well. Cytokines are
currently being used clinically as biological response modifiers for the treatment of various
disorders. The term cytokine is a general term used to describe a large group of proteins but
there are other terms that are commonly used to describe particular kinds of cytokines. These
include: monokines (cytokines produced by mononuclear phagocytic cells), lymphokines
(cytokines produced by activated lymphocytes, especially Th cells), interleukins
(cytokines that acts as mediators between leukocytes), and chemokines (small cytokines primarily
responsible for leukocyte migration).
2) Cytokines function as part of a larger inter-related system of proteins and signaling cascades, the
cytokine network. These are complex interactions in which different cells can respond differently
Page 96
to the same cytokine depending upon other signals received by the cell. Cytokine signaling is
very flexible and can induce both protective and damaging responses.
One cytokine often
influences the synthesis of other cytokines. They can produce cascades, or enhance or
suppress production of other cytokines. In addition, they can often influence the action of other
cytokines. The effects can be: antagonistic, additive, or synergistic.
3) Cytokines are not typically stored as preformed proteins. Rather their synthesis is initiated by
gene transcription and their mRNAs are short lived. They are produced as needed in immune
responses.
Genes encoding cytokines can produce variants through alternative splicing to
yield proteins with slightly different but biologically significant bioactivities.
4) Many individual cytokines are produced by many cell types involved in both the innate and
adaptive immune response. Individual cytokines also act on many cell types (i.e., they are
pleotropic) and in many cases cytokines have similar actions (i.e., they are redundant).
Redundancy is due to the nature of the cytokine receptors.
5)
Receptors for cytokines are heterodimers (sometimes heterotrimers) many of which can be
grouped into families based on common structural features; one subunit is common to all
members of a given family. Some examples are shown in Figure 78 (type 1) and Figure 79 (type
2).
Figure 79: Receptors for cytokines are heterodimers (A)
Page 97
Figure 80: Receptors for cytokines are heterodimers (B)
a) Type 1 cytokine receptors (IL-2R family) are the largest family of cytokine receptors.
This family is divided into three subsets based on common components: IL2R, common ,
and gp130 (Figure 1A). These receptors lack intrinsic protein tyrosine kinase activity. Ligand
(cytokine) binding leads to receptor dimerization and initiation of intracellular signaling.
b) Type 2 cytokine receptors (IFNR family) is denoted by conserved cysteines in the
extracellular domains of the subunits. The extracellular domains also have tandem Ig- like
domains characteristic of this cytokine receptor family. These receptor subunits also have
intrinsic tyrosine kinase activity (denoted by the * in Figure 1B).
c) Chemokine receptors all have seven transmembrane segments linked to GTP-binding proteins.
They are selectively expressed on particular lymphocyte populations and are named based on
the family of chemokines to which they bind; CCR (the CC receptor) binds CC chemokines as its
ligand while the CXCR binds CXC chemokines as its ligand (chemokines naming convention will
be discussed below).
6) Since the subunit common to all members of the family functions in binding cytokine and in
signal transduction, a receptor for one cytokine can often respond to another cytokine in the same
family. Thus, an individual lacking IL-2, for example, is not adversely affected because
other cytokines (IL-15, IL-7, IL-9, etc.) assume its function. Similarly, a mutation in a cytokine
receptor subunit other than the one in common often has little effect. On the other hand, a
mutation in the common subunit has profound effects. For example, a mutation in the gene for
the IL-2R subunit causes human X-linked severe combined immunodeficiency (XSCID)
Page 98
characterized by a complete or nearly complete T and B cell defects.

7) Cytokines bind to specific receptors on target cells with high affinity and the cells that
respond to a cytokine are either: 1) the same cell that secreted cytokine (autocrine); 2) a nearby
cell (paracrine) or 3) a distant cell reached through the circulation (endocrine). Cellular
responses to cytokines are generally slow (hours) because they require new mRNA and protein
synthesis.
8)
Categories of cytokines: Cytokines can be grouped into different categories based on their
functions or their source but it is important to remember that because they can be produced by
many different cells and act on many different cells, any attempt to categorize them will be
subject to limitations.
a) Mediators of the innate immune response: Cytokines that play a major role in the innate immune
system include: TNF-, IL-1, IL-10, IL-12, type I interferons (IFN- and IFN- ), IFN-,
and chemokines.
i) TNF-: Tumor necrosis factor alpha is produced by activated macrophages is response
to microbes, especially the lipopolysaccharide (LPS) of Gram negative bacteria. It is an
important mediator of acute inflammation. It mediates the recruitment of neutrophils and
macrophages to sites of infection by stimulating endothelial cells to produce adhesion molecules
and by producing chemokines which are chemotactic cytokines. TNF- also acts on the
hypothalamus to produce fever and it promotes the production of acute phase proteins.
ii) IL-1: Interleukin 1 is another inflammatory cytokine produced by activated macrophages. Its
effects are similar to that of TNF- and it also helps to activate T cells.
iii) IL-10: Interleukin 10 is produced by activated macrophages and Th2 cells. It is predominantly
an inhibitory cytokine. It inhibits production of IFN- by Th1 cells, which shifts immune
responses toward a Th2 type. It also inhibits cytokine production by activated macrophages
and the expression of class II MHC and co- stimulatory
resulting in a dampening of immune responses.
molecules
on
macrophages,
iv) IL-12: Interleukin 12 is produced by activated macrophages and dendritic cells. It stimulates the
Page 99
production of IFN- and induces the differentiation of Th cells to become Th1 cells. In addition,
it enhances the cytolytic functions of Tc and NK cells.
v) Type I interferons: Type I interferons (IFN- and IFN-) are produced by many cell types and
they function to inhibit viral replication in cells. They also increase expression of class I MHC
molecules on cells making them more susceptible to killing by CTLs. Type I interferons also
activate NK cells.
vi) INF-: Interferon gamma is an important cytokine produced by primarily by Th1 cells,
although it can also be produced by Tc and NK cells to a lesser extent. It has numerous functions
in both the innate and adaptive immune systems as depicted in Figure 2.
Figure 81: Interferon

vii) Chemokines: Chemokines are chemotactic cytokines produced by many kinds of leukocytes
and other cell types. They represent a large family of molecules that function to recruit leukocytes
to sites of infection and play a role in lymphocyte trafficking by determining which cells will
cross the epithelium and where they are directed to go. There are four families of chemokines
based on spacing of conserved cysteine.
Two examples are the -chemokines which
have a CXC structure (two cysteines with a different amino acid in between) and the chemokines which have a CC structure (two neighboring cysteines). Individual chemokines
Page 100
(within the same family) often bind more than one receptor.
b) Mediators of the adaptive immune response: Cytokines that play a major role in the
adaptive immune system include: IL-2, IL-4, IL-5, TGF-, IL-10 and IFN-.
i) IL-2: Interleukin 2 is produced by Th cells, although it can also be produced by Tc cells to a
lesser extent. It is the major growth factor for T cells. It also promotes the growth of B cells and
can activate NK cells and monocytes as depicted in Figure 82. IL-2 acts on T cells in an
autocrine fashion. Activation of T cells results in expression of IL-2R and the production of IL-2.
The IL-2 binds to the IL-R and promotes cell division. When the T cells are no longer being
stimulated by antigen, the IL-2R will eventually decay and the proliferative phase ends Figure 83.
Figure 82: activate NK cells and monocytes, proliferative phase
ii) IL-4: Interleukin 4 is produced by macrophages and Th2 cells. It stimulates the development
of Th2 cells from nave Th cells and it promotes the growth of differentiated Th2 cells resulting
in the production of an antibody response. It also stimulates Ig class switching to the IgE isotype.
iii) IL-5: Interleukin 5 is produced by Th2 cells and it functions to promote the growth and
differentiation of B cells and eosinophils. It also activates mature eosinophils.
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iv) TGF-: Transforming growth factor beta is produced by T cells and many other cell types. It is
primarily an inhibitory cytokine. It inhibits the proliferation of T cells and the activation of
macrophages. It also acts on PMNs and endothelial cells to block the effects of proinflammatory cytokines.
c) Stimulators
of
hematopoesis:
Some
cytokines
stimulate
the
differentiation
of
hematopoietic cells. These include GM-CSF which promotes the differentiation of bone marrow
progenitors, M-CSF, which promotes growth and differentiation of progenitors into monocytes
and macrophages and G-CSF (also known as pluripoietin), which promotes production of PMNs.
d) Interleukin 17: IL-17 is proinflammatory cytokine approximately 150 amino acids long. The
IL-17 family includes sex members which share sequence homology but differential tissue
expression. IL-17 is produced by Th17 cells and its overexpression has been associated with
autoimmune disease including multiple sclerosis, rheumatoid arthritis, and inflammatory bowel
disease.
9)
Cytokine networks: Although the focus of most research and this paper has been on the
production and action of cytokines on cells of the immune system, it is important to
remember that many of them have effects on other cells and organ systems. A schematic diagram
showing some of the interactions in the cytokine network is presented in Figure 5. In fact, the
cytokine network is quite complex and represents a series of overlapping and inter-related
connections amongst cytokines. Within this network, one cytokine may induce or suppress its
own synthesis, induce or suppress the synthesis of other cytokines, induce or suppress synthesis of
cytokine receptors (both its own and other cytokine Rs), and antagonize or synergize with other
cytokines.
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Page 103
Figure 83: Cytokine networks
Page 104

15. Immunoregulation
1) The magnitude
agnitude of an immune response is det
determined
ined by the balance between antigen-driven
antigen
activation of lymphocytes
phocytes and negative regulato
regulatoryy influences that prevent or da
dampen the
response. Regulatory
egulatory mechanisms
mechanis can act at the recognition, activation or effector phases of an
immune response.
2) Regulation in response to Ag has been discussed previously.
a) Recognition of antigen in the abse
absence of co-stimulation resulting in energy,
ener
b) Recognition of antigen with CTLA
CTLA-4 engagement of B7 resulting in doown regulation of T cell
activation,
c) Cytokines with stimulatory
ulatory or inh
inhibitory activities on immune cells,
d) Idiotype/anti-idiotype interacctions leading to stimulation or inhibition of immune responses.
e) Dose and route of Ag exposure can induce diff
differential Th responses(Table 6) which in one case
can protect and in another can tolerate.
Table 6:: Immunoglobulin regulation
Page 105

3) Regulation by antibody
a) Soluble antibody can compete with antigen receptors on B cells
lls and block or prevent B cell
activation (Figure 2A). In this case the regulation is occurring
rring at the recognition level.
b)
In addition antigen antibody complexes

co
xes can bind to Fc receptors on B cells, sending an
inhibitory signal to B cells (Figure 2B). H
Here
re regulation occurs at the activation level.
c) Antibody can also regulate activation (enhance) by maintaining a source of antigen for APC. In
this case, Ab binds Ag form
ming an immune complex
plex which binds and activated the complement
co
system. Complement
ment activation
activ
allows for ligation to the complement
nt rece
receptor on the APC
(Figure 2C).
Figure 84:: Immunoregulation by antibody
Page 106
4) Regulation by cytokines
a) Cytokines are positive or negative regulators. They act at many stages of the immune response,
but their activity is dependent upon the other cytokines present in the microenvironment as well as
receptor expression on effector cells. Cytokines regulate the type and extent of the immune
response generated.
5)
Regulation by regulatory T cells (Tregs): Regulatory T cells (Tregs) are recently described
populations of cells that can regulate immune responses. They do not prevent initial T cell
activation; rather, they inhibit a sustained response and prevent chronic and potentially damaging
responses. They do not have characteristics of Th1, Th2, or Th17 cells but they can suppress both
Th1 and Th2 responses.
a) Naturally occurring Tregs The thymus gives rise to CD4+/CD25+/Foxp3+ cells that function as
Tregs. These Tregs suppress immune responses in a cell contact-dependent manner but the
mechanism of suppression has not been established.
b) Induced Tregs In the periphery some T cells are induced to become Tregs by antigen and either
IL-10 or TGF-. Tregs induced by IL-10 are CD4+/CD25+/Foxp3- and are referred to as Tr1
cells. These cells suppress immune responses by secretion of IL10. Tregs induced by TGF-
are CD4+/CD25+/Foxp3+ and are referred to as induced Tregs. These cells suppress by secretion
of TGF-.
c) CD8+ Tregs Some CD8+ cells can also be induced by antigen and IL-10 to become a Treg cell.
These cells are CD8+/Foxp3+ and they suppress by a cell contact-dependent mechanism or by
secretion of cytokines. These cells have been demonstrated in vitro but it is not known whether
they exist in vivo.
6) Genetic factors influencing immunoregulation
a) MHC-linked genes help control response to infection. Certain HLA haplotypes are associated
Page 107
with individuals who are responders or nonresponder, those who are susceptible or resistant.
b)
Non-MHC genes are also involved in immunoregulation. An example is a gene related to

macrophage activity encoding a transporter protein involved in transport of nitrite (NO2-) into the
phagolysosome, natural resistance-associated macrophage protein-1 (Nramp1). Polymorphisms in
this gene could change the activity of macrophages.
c)
Cytokine, chemokine, and their receptors are involved in immunoregulation as discussed

previously. Polymorphisms in the genes encoding these, in particular the receptors, have been
shown to correlate to susceptibility to infection or generation of autoimmune disease.
16. MHC: genetics and role in transplantation
Histocompatibility (transplantation) antigens: Antigens, on tissues and cells, which determine their
rejection when grafted between two genetically different individuals; Major histocompatibility
(MHC) antigens: Histocompatibility antigens which cause a very strong immune response and are
most important in rejection of organs and tissues; MHC complex: Group of genes on a single
chromosome encoding the MHC antigens; HLA (human leukocyte antigens): MHC antigens of
man (first detected on leukocytes); H-2 antigens: MHC antigens of mouse; Xenograft: Grafts
between members of different species (also known as heterologous or xenogeneic grafts or heterografts); Allograft: Grafts between two members of the same species (also known as allogeneic
grafts or homo-grafts); Isograft/syngeneic : Grafts between members of the same species with
identical genetic makeup (identical twins or inbred animals); Haplotype: a group of genes on a
single chromosome.
Principles of Transplantation:
An immunocompetent host recognizes the foreign antigens on grafted tissues (cells), and mounts
an immune response, which results in rejection (host -vs- graft reaction). On the other hand if an
immunocompromised host is grafted with foreign immunocompetent lymphoid cells, the
immunoreactive T-cells present in the graft recognize the foreign antigens on the host tissue and
cause their damage (graft -vs- host reaction).
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Host-versus-graft-reaction:
The duration of graft survival follows the order, xeno- < allo- < iso- = auto- graft. The time of
rejection also depends on the antigenic disparity between the donors and recipient. While the
MHC antigens are the major contributors in rejection, the minor histocompatibility antigens
also play a significant role. Rejection due to disparity in several minor histocompatibility
antigens may be as quick or quicker than rejection mediated by an MHC antigen. Like in other
immune responses, there is immunological memory and secondary response in graft rejection.
Thus, once a graft is rejected by a recipient, a second graft from the same donor, or a donor
with the same histocompatibility antigens, will be rejected in a much shorter time.
Graft-versus-host (GVH) reaction:

Histoincompatible lymphoid cells when injected into an immunosuppressed host are readily
accepted. However, the immunocompetent T lymphocytes found in the grafted cells recognize
the alloantigens found on the host and proliferate and progressively cause damage to the host
tissues and cells. This condition is known as graft-versus-host (GVH) disease and is often fatal.
Common manifestations of GVH reaction are diarrhea, erythema, weight loss, malaise, fever,
joint pains, etc. and ultimately death.
Figure 85: The human MHC gene complex
Page 109
The MHC Gene Complex:

The MHC complex contains a number of genes, which control several antigens,most of which
influence allograft rejection. These antigens (and their genes) can be divided into three major
classes: class I, class II and class III. The class I and class II antigens are expressed on cells and
tissues whereas as class III antigens are associated with proteins in serum and other body-fluids
(e.g.C4, C2, factor B, TNF). While antigens from class I and class II gene products play a critical
role in transplantation, those from class III gene products have no direct role in immune
responses that determine graft survival.
Human MHC:
The human MHC is located on chromosome 6.
Class I MHC:
The class I gene complex contains three major loci of highest significance, B, C and A and some
undefined loci of less significance (Figure 1). Each these loci codes for a polypeptide, -chain
that contains antigenic determinants that are polymorphic (has many alleles). Each -chain
associates with a -2 microglobulin molecule (-chain), encoded by a gene outside the MHC
complex. The --chain complex is expressed on the cell surface as the class-I MHC antigen.
Without a functional -2 microglobulin chain, the class I antigen will not be expressed on the
cells surface. Individuals with defective a -2 microglobulin gene do not express any class I
antigen and hence they have a deficiency of cytotoxic T cells.
Class II MHC:
The class II gene complex also contains at least three loci, DP, DQ and DR; each of these loci
codes for one - and a variable number of -chain polypeptides which associate together to form
the class II antigens. Like the class I antigens, the class II antigens are also polymorphic. The
DR locus contains more than one, possibly 4, functional -chain genes.
MHC Polymorphism: MHC complex is the most polymorphic in the genome. This means that
there is an astonishing allelic diversity found within MHC. In humans, the most conspicuouslydiverse loci, HLA-A, HLA-B, and HLA-DRB1, have roughly 250, 500, and 300 known alleles
respectively. This helps protect the species from extinction that could result from infections and
Page 110
other diseases. However, it is for this very reason, it is extremely difficult to match the donor and
the recipient.
Mouse MHC:
The mouse MHC is located on chromosome 17.
Class I MHC:
It consists of two major loci, K and D. Unlike the human MHC, the mouse class I gene complex
loci are not together but they are separated by class II and class III genes (Figure 87).
Figure 86: The mouse MHC complex

Class II MHC:
The class II gene complex of mouse contains two loci, A and E each of which code for one and one - chain polypeptide, which form one class II molecule. The mouse class II gene
complex is also known as the I-region and the genes in this complex are referred to as Ir
(immune response) genes since they determine the magnitude of immune responsiveness of
different mouse strains to certain antigens. Products of A and E loci are also termed IA and IE
antigens, collectively known as Ia antigens.
MHC
ANTIGENS:
Nomenclature:
HLA specificities are identified by a letter for locus and a number (A1, B5, etc.), and the
haplotypes are identified by individual specificities (e.g., A1, B7, Cw4, DP5, DQ10 DR8).
Specificities which are defined by genomic analysis (PCR), are named with a letter for the locus
and a four digit number (e.g. A0101, B0701, C0401, etc.) Specificities of Mouse MHC (H-2) are
identified by a number.Each strain is homozygous and has a unique haplotype. The MHC
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haplotype in these strains is designated by a small letter (a, b, d, k, q, s, etc.). For example,
the MHC haplotype of Balb/c, an inbred strain of mouse, is H2d.
Inheritance:
MHC genes are inherited as a group (haplotype), one from each parent. Thus, a heterozygous
human inherits one paternal and one maternal haplotype, each containing three class-I (B, C and
A) and three class II (DP, DQ and DR) loci. A heterozygous individual will therefore inherit a
maximum of 6 class I specificities (Figure 3: top). Similarly, the individual will also inherit DP
and DQ genes and express both parental antigens. Since the class II MHC molecule consists of
two chains ( and ), with some antigenic determinants (specificities) on each chain, and DR
- and -chains can associate in cis (both from the same parent) or trans (one from each parent)
combination, an individual can have additional DR specificities (Figure 3: bottom). Also, there
are more than one functional DR -chain genes (not shown in the figure). Hence, many DR
specificities can be found in any one individual.
Figure 87: Co-dominant expression of MHC antigens

Crossover:
Haplotypes, normally, are inherited intact and hence antigens encoded by different loci are
Page 112
inherited together (e.g., A2; B27; Cw2; DPw6; DQw9; DRw2). However, on occasions, there is
crossing over between two parental chromosomes, thereby resulting in new recombinant
haplotypes. Thus any specificity encoded by one locus, may combine with specificities from
other loci. This results in a vast heterogeneity in the MHC make-up in a given population.
MHC antigen expression on cells:
MHC antigens are expressed on the cell surface in a co-dominant manner: products of both
parental genes are found on the same cells. However, not all cells express both class I and class
II antigens. While class I antigens are expressed on all nucleated cells and platelets (and red
blood cells in the mouse), the expression of class II antigens is more selective. It is expressed
on B lymphocytes, a proportion of macrophages and monocytes, skin associated (Langerhans)
cells, dendritic cells and occasionally on other cells.
MHC detection by serological test:
The MHC class I antigens are detected by serological assays (Ab and C). Tissue typing sera for
the HLA were obtained, in the past, from multiparous women who were exposed to the child=s
paternal antigens during the parturition and subsequently developed antibodies to these antigens.
More recently they are being produced by the monoclonal antibody technology. With most
laboratories switching to PCR for tissue typing, the use of serology is rapidly diminishing.
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Figure 88: Activation of CTL and Mechanism of Allograft Destruction
MHC detection by mixed leukocyte reaction (MLR):

It has been observed that lymphocytes from one donor, when cultured with lymphocytes from an
unrelated donor, are stimulated to proliferate. It has been established that this proliferation is
primarily due to a disparity in the class II MHC (DR) antigens and T cells of one individual
interact with allogeneic class-II MHC antigen bearing cells (B cells, dendritic cells, langerhans
cells, etc.). This reactivity was termed mixed leukocyte reaction (MLR) and has been used for
studying the degree of histocompatibility. In this test, the test lymphocytes (responder cells)are
mixed with irradiated or mitomycin-C treated leukocytes from the recipient, containing Blymphocytes and monocytes (stimulator cells).
The cells are culturedfor 4-6 days. The responder T-cells will recognize the foreign class II
antigens found on the donor and undergo transformation (DNA synthesis and enlargement:
blastogenesis) and proliferation (mitogenesis). The T cells that respond to foreign class II
antigens are typically CD4+ TH-1 type cells. These changes are recorded by the addition of
radioactive (tritiated, 3H) thymidine into the culture and monitoring its incorporation into DNA.
Generation of cytotoxic T lymphocytes
Another consequence of the MHC antigen and T cell interaction is the induction of cytotoxic Tlymphocytes. When T-lymphocytes are cultured in the presence of allogeneic lymphocytes, in
addition to undergoing mitosis (MLR), they also become cytotoxic to cells of the type that
stimulated MLR (Figure 4). Thus, T-lymphocytes of 'x' haplotype cultured over 5-7 days with B
lymphocytes of 'y' haplotype will undergo mitosis and the surviving T-lymphocytes become
cytotoxic to cells of the 'y' haplotype. The cytotoxic T-lymphocytes (CTL) primarily recognize
class-I antigens and are CD8+.
Allograft Rejection
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The clinical significance of the MHC is realized in organ transplantation. Cells and tissues are
routinely transplanted as a treatment for a number of diseases. However, reaction of the host
against alloantigens of the graft (HVG) results in its rejection and is the major obstacle in organ
transplantation. The rejection time of a graft may vary with the antigenic nature of the graft and
the immune status of the host and is determined by the immune mechanisms involved (Figure 5).
Hyper-acute rejection:
This occurs in instances when the recipient has preformed high titer antibodies. A graft may
show signs of rejection within minutes to hours due to immediate reaction of antibodies and
complement.
Accelerated (2nd set; secondary) rejection:
Transplantation of a second graft, which shares a significant number of antigenic determinants
with the first one, results in a rapid (2-5 days) rejection. This is due to presence of Tlymphocytes sensitized during the 1st graft rejection. Accelerated rejection is mediated by
immediate production of lymphokines, activation of monocytes and macrophages and induction
of cytotoxic lymphocytes.
Acute (1st set; primary) rejection:
The normal reaction, which follows the first grafting of a foreign transplant, takes 1-3 weeks. This
is known as acute rejection and is mediated by T lymphocytes sensitized to class-I and class-II
antigens of the allograft, lymphokines, activated of monocytes and macrophages.
Chronic rejection:
Some grafts may survive for months or even years, but suddenly exhibit symptoms of rejection.
This is referred to as chronic rejection, the mechanism of which is not entirely clear. The
hypotheses are that this may be due infection, causes which led to failure of the first organ, loss of
tolerance induced by the graft, etc.
Fetus as an Allograft: The fetus in an out-bred mammalian species bears antigens derived from
both the father and the mother. Thus, truly, the fetus is an allograft and the mother should
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normally recognize the fetus as foreign and reject the fetus. Nonetheless, such rejections seldom
occur. Thus, mammals have adapted in a way that allows implantation of their embryos in the
mother's womb and their subsequent survival. There are multiple mechanisms that play a role, of
which the most important being the unique structure and function of placenta.
Immunologically privileged sites and tissues: There are certain locations in the body in which
allografts are not readily rejected. These include the brain, anterior chamber of the eye, testis,
renal tubule, uterus, etc. This stems from the fact that such sites may lack of good lymphatic
drainage. Also, such tissues may express molecules such as Fas ligand that kills any immune cell
that may come in contact with these tissues. Additionally, such tissues, may have other immune
suppressor mechanisms.
Similarly, there are some tissues that can be transplanted without
matching and without being rejected. Such tissues are called immunologically privileged tissues.
Corneal graft is an excellent example that enjoys the highest success rate of any form of organ
transplantation. The low incidence of graft rejection is impressive despite the fact that HLA
antigen matching of donor and recipient is not normally performed. There are many explanations
as to why such grafts are accepted.
The avascularity of the graft bed prevents corneal
alloantigens from reaching the regional lymphoid tissues. Also, the corneal antigens may be
masked. Together, such mechanisms fail to activate the immune system of the recipient.
Procedures to Enhance Graft Survival
In clinical practice, the most successful transplantation programs have been with kidneys and
corneas. However, other organs are being transplanted with increasing frequency and success.
The success in these programs has been due to a better understanding of immunological
mechanisms, definition of
immunosuppressive agents.
MHC
antigens
and
development
of
more
effective
Donor selection:
Based on extensive experiences with renal transplants certain guidelines can be followed in
donor selection and recipient preparation for most organ transplants. The most important in
donor selection is the MHC identity with the recipient; an identical twin is the ideal donor.
Grafts from HLA-matched sibling have 95-100% chance of success. One haplotype-identical
parent or sibling must match at HLA D region. A two-haplotype-distinct donor with reasonable
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match for D-region antigen can also be used. Organ from two or one DR matched cadaver has
been used also with some success. In every case, an ABO-compatibility is essential.
Recipient preparation:
The recipient must be screened for donor-specific anti-HLA antibodies and be negative, must be
infection free and must not be hypertensive. One to five transfusions of 100-200 ml whole blood
from the donor at 1-2 week intervals improves the graft survival and is practiced when possible.
Immunosuppression:
Immunosuppressive therapy is most essential part of allo-transplantation. The most recent and
effective family of agents is cyclosporine, tacrolimus (formerly, FK-506; Prograft) and
rapamycin (Rapmune). Cyclosporine and tacrolimus inhibits IL-2 synthesis following Agreceptor binding whereas rapamycin interferes with signal transduction following IL2-IL2R
interaction. Thus, all these three agents block T cell proliferation in response to antigen. Other
chemical agents used to prevent graft rejection and their modes of action have been listed in Table
2. Whole body irradiation is used in leukemia patients before bone marrow transplantation.
Antisera against T cells (anti-thymocyte globulin: ATG) or their surface antigens (CD3, CD4,
CD45on activated T-cells, CD25: IL-2 receptors) are being used also to achieve
immunosuppression.
Strategies for bone marrow transplantation:
In bone marrow transplantation, the most crucial factor in donor selection is class II MHC
compatibility. Once again, an identical twin is the ideal donor. From poorly matched grafts, T
lymphocytes can be removed using monoclonal antibodies.
The recipient must be
immunosuppressed. Malignant cells must be eliminated from the recipient blood (in case of
blood-borne malignancies). Methotrexate, cyclosporin and prednisone are often used to control
GVH disease.
Other grafts:
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Corneal grafts do not contain D region antigens and consequently survival is frequent. Small
grafts are better and their survival is improved by corticosteroid use.
Skin allograft have very poor success rate and immunosuppressive therapy is relatively
ineffective. Nevertheless, they are often used to provide a temporary covering to promote healing
in severe skin damage. Indeed, there will be no rejection, if the host and donor are perfectly
matched (identical twins) or the recipient is tolerant to the donor MHC antigens (bone marrow
chimeras).
17. Hypersensitivity reactio
reactions
Hypersensitivity refers to excessive undesirable (damaging, discomfort producing and sometimes
fatal) reactions produced by the normal immune system. Hypersensitivity reactions require a presensitized (immune) state of the host. Hypersensitivity reactions can be divided into four types:
type I, type II, type III and type IV, based on the mechanisms involved and time taken for the
reaction. Frequently, a particular clinical condition (disease) may involve more than one type of
reaction.
Type I Hypersensitiv
Hypersensitivity
It is also known as immediate or anaphylactic hypersensitivity. The reaction may involve skin
(urticaria and eczema), eyes (conjunctivitis), nasopharynx (rhinorrhea, rhinitis), bronchopulmonary
tissues (asthma) and gastrointestinal tract (gastroenteritis).
inconvenience to death.
Type I hypersensitivity is mediated by IgE.
The reaction may cause from minor
The primary cellular component in this
hypersensitivity is mast cell or basophil. The reaction is amplified and/or modified by platelets,
neutrophils and eosinophils. A biopsy of the reaction site demonstrates mainly mast cells and
eosinophils. The mechanism of reaction involves preferential production of IgE, in response to
certain antigens, often called allergens (Figure 1). The precise mechanism as to why some
individuals are more prone to type-I hypersensitivity is not clear. However, it has been shown
that such individuals preferentially produce more of TH2 cells that secrete IL-4, IL-5 and IL-13
which in turn favor IgE class switch. IgE has very high affinity for its receptor (Fc; CD23) on
mast cells and basophils. A subsequent exposure to the same allergen cross links the cellbound IgE and triggers the release of various pharmacologically active substances (Figure 1).
Page 118
Cross-linking of IgE Fc-receptor is important in mast cell triggering. Mast cell degranulation is
preceded by increased Ca++ influx, which is a crucial process; ionophores which increase
cytoplasmic Ca++ also promote degranulation of mast cells, whereas, agents which deplete
cytoplasmic Ca++ suppress degranulation.
The agents released from mast cells and their effects are listed in Table 1. Mast cells may
be triggered by other stimuli such as exercise, emotional stress, chemicals (e.g., photographic
developing medium, calcium ionophores, codeine, etc.), anaphylotoxins (e.g., C4a, C3a, C5a,
etc.). These reactions mediated by agents without IgE-allergen interaction are not typical
hypersensitivity reactions, although they produce the same symptoms.
The reaction is amplified by PAF (platelet activation factor) that causes platelet aggregation and
release of histamine, heparin and vasoactive amines. Eosinophil chemotactic factor of anaphylaxis
(ECF-A) and neutrophil chemotactic factors attract eosinophils and neutrophils, respectively,
which release various hydrolytic enzymes that cause necrosis. Eosinophil may also control the
local reaction by releasing arylsulphatase, histaminase, phospholipase-D and prostaglandin-E,
although this role of eosinophils is now in question.
Cyclic nucleotides appear to play a significant role in the modulation of immediate
hypersensitivity reaction, although their exact function is ill understood. Substances which alter
cAMP and cGMP levels significantly alter the allergic symptoms. Thus, substances that increase
intracellular cAMP seem to relieve allergic symptoms, particularly broncho-pulmonary ones, and
are used therapeutically. Conversely, agents that decrease cAMP or stimulate cGMP aggravate
these allergic conditions.
Diagnostic tests for immediate hypersensitivity include skin (prick and intradermal) tests resulting
in wheal and flare reaction, measurement of total IgE and specific IgE antibodies against the
suspected allergens. Total IgE and specific IgE antibodies are measured by a modification of
enzyme immunoassay (ELISA). Increased IgE levels are indicative of atopic condition, although
IgE may be elevated in some non atopic diseases (e.g., myelomas, helminthic infection, etc.).
Page 119
There appears to be a genetic predisposition for atopic diseases and there is evidence for
HLA (A2) association.
Symptomatic treatment is achieved with antihistamines that block histamine receptors.
Chromolyn sodium inhibits mast cell degranulation, probably, by inhibiting Ca++ influx. Late
onset allergic symptoms, particularly bronchoconstriction which is mediated by leukotrienes are
treated with leukotriene receptor blockers (Singulair, Accolate, Lukast) or inhibitors of
cyclooxygenase pathway (Zileutoin).
Symptomatic, although short-term, relief from
bronchoconstriction is provided by bronchodilators (inhalants) such as isoproterenol derivatives
(Terbutaline, Albuterol). Thophylline elevates cAMP by inhibiting cAMP-phosphodiesterase and
inhibits intracellular Ca++ release is also used to relieve bronchopulmonary symptoms.
IgG antibodies against the Fc portions of IgE that binds to mast cells has been approved for
treatment of certain allergies, as it can block mast cell sensitization. Hyposensitization
(immunotherapy or desensitization) is another treatment modality which is successful in a
number of allergies, particularly to pollen. The mechanism is not clear, but there is a correlation
between appearance of IgG (blocking) antibodies and relief from symptoms. Suppressor T cells
that specifically inhibit IgE antibodies may play a role.
Type II Hypersensitivity
It is also known as cytotoxic hypersensitivity and may affect a variety of organs and tissues.
The antigens are normally endogenous, although exogenous chemicals (haptens) that can attach to
cell membranes can also lead to type II hypersensitivity. Drug-induced hemolytic anemia,
granulocytopenia and thrombocytopenia are such examples. The reaction time is minutes to
hours. It is mediated, primarily, by antibodies of IgM or IgG class and complement (Figure 92).
Phagocytes a n d K ( killer) cells m a y a l s o play a role.
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Figure 89:
89: types of hypersensitivity reaction
The lesion contains antibody, complement and neutrophils. Diagnostic tests include detection of
circulating antibody against tissues involved and the presence of antibody and complement in the
lesion (biopsy) by immunofluorescence. The staining pattern is normally smooth and linear, such
as that seen in Goodpastures nephritis (renal and lung basement membrane) and pemphigus
(skin intercellular protein, desmosome).
Type III Hy
Hypersensitivity
It is also known as immune complex hypersensitivity. The reaction may be general (e.g., serum
sickness) or may involve individual organs including skin (e.g., systemic lupus erythematosus,
Arthus reaction), kidneys (e.g., lupus nephritis), lungs (e.g., aspergillosis), blood vessels (e.g.,
polyarteritis), joints (e.g., rheumatoid arthritis) or other organs.
pathogenic mechanism of diseases caused by many microorganisms.
This reaction may be the
The reaction may take 3-10 hours after exposure to the antigen (as in Arthus reaction). It is
mediated by soluble immune complexes. They are mostly of IgG class, although IgM may also be
involved. The antigen may be exogenous (chronic bacterial, viral or parasitic infections), or
endogenous (non-organ specific autoimmunity: e.g., systemic lupus eythematosus-SLE).
The
antigen is soluble and not attached to the organ involved. Primary components are soluble
immune complexes and complement (C3a, 4a and 5a). The damage is caused by platelets and
neutrophils (Figure 3).
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The lesion contains primarily neutrophils and deposits of immune complexes and
complement. Macrophages infiltrating in later stages may be involved in the healing process.
Figure 90: Mechanism of damage in type-III hypersensitivity

The affinity of antibody and size of immune complexes are important in production of disease and
determining the tissue involved. Diagnosis involves examination of tissue biopsies for deposits of
Ig and complement by immunofluorescence.
The immunofluorescent staining in type III
hypersensitivity is granular (as opposed to linear in type II: Goodpasture). Presence of immune
complexes in serum and depletion in complement level are also diagnostic. Treatment includes
anti-inflammatory agents.
Type IV Hypersensitivity
It is also known as cell mediated or delayed type hypersensitivity. The classical example of this
hypersensitivity
is
tuberculin (Montoux) reaction that peaks 48 hours after the injection of
antigen (PPD or old tuberculin). The lesion is characterized by induration and erythema.
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Figure 91: Mechanisms of damage in delayed hypersensitivity
Type IV hypersensitivity is involved in the pathogenesis of many autoimmune and infectious

diseases (tuberculosis, leprosy, blastomycosis, histoplasmosis, toxoplasmosis, leishmaniasis, etc.)
and
granulomas
due to infections and foreign antigens. Another form of delayed
hypersensitivity is contact dermatitis (poison ivy, chemicals, heavy metals, etc.) in which the
lesions are more papular.
Type IV hypersensitivity can be classified into three categories
depending on the time of onset and clinical and histological presentation (Table 7).
Mechanisms of damage in delayed hypersensitivity include T lymphocytes and monocytes and/or
macrophages.
The pathogenesis is triggered primarily by helper T (TH1) cells that secrete
cytokines that activate and recruit macrophages, which cause the bulk of the damage (Figure 3).
The delayed hypersensitivity lesions mainly contain monocytes and T cells.
Major lymphokines involved in delayed hypersensitivity reaction include monocyte chemotactic

factor, interleukin-2, interferon-, TNF , etc. Diagnostic tests in vivo include delayed
cutaneous reaction (e.g. Montoux test) and patch test (for contact dermatitis). In vitro tests
for delayed hypersensitivity include mitogenic response, lympho-cytotoxicity and IL-2
production.
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17. Tolerance and Autoimmunity

17.1. Tolerance Introduction:
Tolerance refers to the specific immunological non-reactivity to an antigen resulting from
a previous exposure to the same antigen. While the most important form of tolerance is nonreactivity to self antigens, it is possible to induce tolerance to non-self (foreign) antigens. When
an antigen induces tolerance, it is termed tolerogen.
Tolerance to self antigens: We normally do not mount a strong immune response against our
own (self) antigens, a phenomenon called self-tolerance.
When the immune system
recognizes a self antigen and mounts a strong response against it, autoimmune disease develops.
Nonetheless, the immune system has to recognize self-MHC to mount a response against a
foreign antigen. Thus, the immune system is constantly challenged to discriminate self vs nonself and mediate the right response.
Induction of tolerance to non-self : Tolerance can also be induced to non-self (foreign) antigens
by modifying the antigen, by injecting the antigen through specific routes such as oral,
administering the antigen when the immune system is developing, etc. Certain bacteria and
viruses have devised clever ways to induce tolerance so that the host does not kill these microbes.
Ex:
Patients with lepromatous type of leprosy do not mount an immune response against
Mycobacterium leprae.
Tolerance to tissues and cells:
Tolerance to tissue and cell antigens can be induced by injection of hemopoietic (stem) cells in
neonatal or severely immunocompromised (by lethal irradiation or drug treatment) animals. Also,
grafting of allogeneic bone marrow (or thymus) in early life results in tolerance to the donor type
cells and tissues. Such animals are known as chimeras. These findings are of significant
importance in bone marrow grafting.
Immunologic features of tolerance:

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Tolerance is different from non-specific immunosuppression, and immunodeficiency. It is an

active antigen dependent process in response to the antigen. Like immune response, tolerance is
specific and like immunological memory, it can exist in T-cell, B cells or both and like
immunological memory, tolerance at the T cell level is longer lasting than tolerance at the B cell
level.
Induction of tolerance in T cells is easier and requires relatively smaller amounts of tolerogen
than tolerance in B cells. Maintenance of immunological tolerance requires persistence of antigen.
Tolerance can be broken naturally (as in autoimmune diseases) or artificially (as shown in
experimental animals, by x-irradiation, certain drug treatments and by exposure to cross reactive
antigens).
Tolerance may be induced to all epitopes or only some epitopes on an antigen and tolerance
to a single antigen may exist at B cell level or T cells level or at both levels.
Mechanisms of tolerance induction:
The exact mechanism of induction and maintenance of tolerance is not fully understood.
Experimental data, however, point to several possibilities.
Clonal deletion: T and B lymphocytes during development come across self antigens and such
cells undergo clonal deletion through a process known as apoptosis or programmed cell death.
For example, T cells that develop in the thymus first express neither CD4 nor CD8. Such cells
next acquire both CD4 and CD8 called double-positive cells and express low levels of
TCR. Such cells undergo positive selection after interacting with class I or class II MHC
molecules expressed on cortical epithelium.
During this process, cells with low affinity for
MHC are positively selected. Unselected cells die by apoptosis, a process called death by
neglect. Next, the cells loose either CD4 or CD8. Such T cells then encounter selfpeptides presented by self MHC molecules expressed on dendritic cells. Those T cells with high
affinity receptors for MHC + self-peptide undergo clonal deletion also called negative selection
through induction of apoptosis. Any disturbance in this process can lead to escape of autoreactive T-cells that can trigger autoimmune disease. Likewise, differentiating early B cells
when they encounter self-antigen, cell associated or soluble, undergo deletion. Thus, clonal
deletion plays a key role in ensuring tolerance to self antigen.
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Peripheral tolerance: The clonal deletion is not a fool proof system and often T and B cells fail
to undergo deletion and therefore such cells can potentially cause autoimmune disease once they
reach the peripheral lymphoid organs. Thus, the immune system has devised several additional
check points so that tolerance can be maintained.
Activation-induced cell death: T cells upon activation not only produce cytokines or carryout
their effector functions but also die through programmed cell death or apoptosis. In this process,
the death receptor (Fas) and its ligand (FasL) play a crucial role. Thus, normal T cells express
Fas but not FasL.
Upon activation, T cells express FasL which binds to Fas and triggers
apoptosis by activation of caspase-8. The importance of Fas and FasL is clearly demonstrated by
the observation that mice with mutations in Fas (lpr mutation) or FasL (gld mutation) develop
severe lymphoproliferative and autoimmune disease and die within 6 months while normal mice
live up to 2 years.
Similar mutations in these apoptotic genes in humans leads to a
lymphoproliferative disease called autoimmune lymphoproliferative syndrome (ALPS).
Clonal anergy: Auto-reactive T cells when exposed to antigenic peptides on antigen presenting
cells (APC) that do not possess the co-stimulatory molecules CD80 (B7-1) or CD86 (B7-2)
become anergic (nonresponsive) to the antigen. Also, while activation of T cells through CD28
triggers IL-2 production, activation of CTLA4 leads to inhibition of IL-2 production and anergy.
Also, B cells when exposed to large amounts of soluble antigen down-regulate their surface IgM
and become anergic. These cells also up-regulate the Fas molecules on their surface. An
interaction of these B cells with Fas-ligand bearing T cells results in their death via apoptosis.
Clonal ignorance: T cells reactive to self-antigen not represented in the thymus will mature and
migrate to the periphery, but they may never encounter the appropriate antigen because it is
sequestered in inaccessible tissues. Such cells may die out for lack of stimulus. Auto-reactive B
cells, that escape deletion, may not find the antigen or the specific T-cell help and thus not be
activated and die out.
Anti-idiotype antibody: These are antibodies that are produced against the specific idiotypes of
other antibodies.
Anti-idiotypic antibodies are produced during the process of
tolerization and have been demonstrated in tolerant animals. These antibodies may prevent the
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B cell receptor from interacting with the antigen.

Regulatory T cells (Formerly called suppressor cells): Recently, a distinct population of T cells
has been discovered called regulatory T cells. Regulatory T cells come in many flavors,
but the most well characterized include those that express CD4+ and CD25+. Because
activated normal CD4 T cells also express CD25, it was difficult to distinguish regulatory T
cells and activated T cells. The latest research suggests that regulatory T cells are defined by
expression of the forkhead family transcription factor Foxp3. Expression of Foxp3 is required for
regulatory T cell development and function. The precise mechanism/s through which regulatory T
cells suppress other T cell function is not clear. One of the mechanisms include the production
of immunosuppressive cytokines such as TGF- and IL-10. Genetic mutations in Foxp3 in
humans leads to development of a severe and rapidly fatal autoimmune disorder known as
Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX)syndrome. This
disease provides the most striking evidence that regulatory T cells play a critical role in preventing
autoimmune disease.
Table 7: Factors which determine induction of immune response or tolerance following challenge
with antigen.
determinant
favor immune response
favor tolerance
soluble, aggregate-free, relatively
smaller, less complex molecules, Ag
physical form of antigen
large, aggregated, complex
molecules;
route of Ag administration sub-cutaneous or intramuscular
not processed by APC or processed

oral or sometimes intravenous
very large (or sometime very small)
dose of antigen
optimal dose
dose
age of responding animal
older and immunologically
Newborn (mice), immunologically
differentiation state of
cells
fully differentiated cells;
relatively undifferentiated: B cells

memory T and memory B cells
with only IgM (no IgD), T cells (e.g.

cells in thymic cortex)
17.2. Autoimmunity
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Definition:
Autoimmunity can be defined as breakdown of mechanisms responsible for self-tolerance and
induction of an immune response against components of the self. Such an immune response may
not always be harmful (e.g., anti-idiotype antibodies or recognition of self-MHC molecules).
However, in numerous (autoimmune) diseases it is well recognized that products of the immune
system cause severe damage to the self.
Effector mechanisms in autoimmune diseases:
Both antibodies and effector T cells and their products can be involved in the damage in
autoimmune diseases.
General classification:
Autoimmune diseases are generally classified on the basis of the organ or tissue involved. These
diseases may fall in an organ-specific category in which the immune response is directed against
antigen(s) associated with the target organ being damaged or a non-organ-specific (also sometimes
referred to as systemic) in which the antibody is directed against an antigen or many antigens not
associated with the target organ and the disease is seen throughout the body (Table 1). The
antigen involved, in most autoimmune diseases is evident from the name of the disease (Table 1).
Genetiic pred
Genet
predisposition for autoimmu
autoimmunity:
Studies in mice and observations in humans suggest a genetic predisposition for autoimmune
diseases. Association between certain HLA types and autoimmune diseases has been noted
(HLA: B8, B27, DR2, DR3, DR4, DR5 etc.).
Etiology of autoimmunity disease:
The exact etiology of autoimmune diseases is not known. However, various theories have been
offered. These include sequestered antigen, escape of auto-reactive clones, loss of Regulatory T
cells, cross-reactive antigens including exogenous antigens (pathogens) and altered self antigens
(chemical and viral infections).
Sequestered antigen: Lymphoid cells may not be exposed to some self-antigens during their
differentiation, because they may be late-developing antigens or may be confined to specialized
organs (e.g., testes, brain, eye, etc.). A release of antigens from these organs, resulting from
accidental traumatic injury or surgery, can result in the stimulation of an immune response and
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initiation of an autoimmune disease.

Escape of auto-reactive clones: The negative selection in the thymus may not be fully
functional to eliminate self reactive cells. Not all self antigens may be represented in the thymus
or certain antigens may not be properly processed and presented.
Lack of regulatory T cells: There are fewer regulatory T-cells in many autoimmune diseases.
Cross reactive antigens: Antigens on certain pathogens may have determinants which cross react
with self antigens and an immune response against these determinants may lead to effector cells
or antibodies against tissue antigens. Post streptococcal nephritis and carditis, anticardiolipin
antibodies during syphilis and association between Klebsiella and ankylosing spondylitis are
examples of such cross reactivity.
Diagnosis:
Diagnosis of autoimmune diseases is based on symptoms and detection of antibodies (and/or very
rarely T cells) reactive against antigens of tissues and cells involved. Antibodies against
cell/tissue-associated antigens are detected by immunofluorescence. Antibodies against soluble
antigens are normally detected by ELISA or radioimmunoassay (see table above). In some
cases, a biological /biochemical assay may be used (e.g., Graves diseases, pernicious anemia).
Treatment:
The goals of treatment of autoimmune disorders are to reduce symptoms and control the
autoimmune response while maintaining the body's ability to fight infections. Treatments vary
widely and depend on the specific disease and symptoms:
Anti-inflammatory (corticosteroid)
and immunosuppressive drug therapy (such as cyclophosphamide, azathioprine, cyclosporine )

is the present method of treating autoimmune diseases. Extensive research is being carried out
to develop innovative treatments which include: anti-TNF alpha therapy against arthritis, feeding
antigen orally to trigger tolerance, anti-idiotype antibodies, antigen peptides, anti-IL2 receptor
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antibodies, anti-CD4 antibodies, anti-TCR antibodies, etc.
Models of autoimmune diseases:

There are a number of experimental and natural animal models for the study of autoimmune
diseases. These experimental models include experimental allergic encephalomyelitis(EAE)
which
mimics
Multiple Sclerosis, experimental thyroiditis, adjuvant induced arthritis, etc.
Naturally occurring models of autoimmune diseases include hemolytic anemia in NZB mice,
systemic lupus erythematosus in NZB/NZW (BW), BXSB and MRL lpr/lpr mice and diabetes in
NOD (non-obese diabetic) mice.
18. Tumor Immunology
Malignant Transformation:
The proliferation of normal cells is carefully regulated. However, such cells when exposed to
chemical carcinogens, irradiation and certain viruses may undergo mutations leading to their
transformation into cells that are capable of uncontrolled growth, producing a tumor or neoplasm.
A tumor may be
1) Benign, if it is not capable of indefinite growth and the host survives.
2) Malignant, if the tumor continues to grow indefinitely and spreads (metastasizes), eventually
killing the host.
This uncontrolled growth may be due to upregulation of oncogenes (cancer-inducing genes)
and/or downregulation of tumor suppressor genes (that normally inhibit tumor growth often by
inducing cell death).
Evidence for existence of an immune response against tumors
The following criteria serve as evidence that tumors can elicit an immune response.
1.
Certain tumors regress spontaneously (e.g., melanomas, neuroblastomas).suggesting an

immunological response.
2. Tumors that have severe mononuclear cell infiltration have a better prognosis than those that
lack it.
3. Some tumor metastases regress after removal of primary tumor which reduces the tumor
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load, thereby inducing the immune system to kill the residual tumor..
4. Although chemotherapy leads to rejection of a large number of tumor cells, the few tumor cells
that evade the action of the drugs can outgrow and kill the host. However, the immune system
may be able to mount an attack against the few tumor cells that are spared by the
chemotherapeutic agent.
5. There is an increased incidence of malignancies in immunodeficient patients such as AIDS
patients who are susceptible to Kaposi sarcoma and transplant patients who are susceptible to
Epstein Barr virus (EBV)- induced lymphoma.
6.
Tumor-specific antibodies and T lymphocytes (detected in cytotoxicity and proliferative
response assays) have been observed in patients with tumors.

7. The young and the old population have an increased incidence of tumors. These members of the
population often have an immune system that is compromised.
8. Hosts can be specifically immunized against various types of tumors demonstrating tumor Ags
can elicit an immune response.
Tumor antigens
Tumorigenesis may lead to expression of new antigens or alteration in existing antigens
that are found on normal cells. These antigens may include membrane receptors,
regulators of cell cycle and apoptosis, or molecules involved in signal transduction
pathways. There are 2 main types of tumor antigens.
1.
Tumor-specific transplantation antigens (TSTA) which are unique to tumor cells and not
expressed on normal cells. They are responsible for rejection of the tumor.
2. Tumor associated transplantation antigens (TATA) that are expressed by tumor cells and
normal cells.
Although chemical- , UV- or virus-induced tumors express neo-antigens, majority of the tumors are
often weakly immunogenic or non-immunogenic. In most cases, tumor-specific transplantation
Ags cannot be identified easily. Also, some of these antigens may be secreted while others include
membrane-associated molecules.
Tumor associated transplantation antigens (TATA)

The majority of tumor Ags are the tumor associated transplantation antigens (TATA). They may
be expressed at higher levels on tumor cells when compared to normal cells. Alternatively, they
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may be expressed only during development of cells and lost during adult life but re-expressed in
tumors. These include the tumor-associated developmental Ags (TADA) and tumor-associated
viral Ags (TAVA).
Tumor-associated developmental Ags (TADA) or Onco-fetal antigens
These include alpha-fetoprotein (AFP) and carcino-embryonic antigen (CEA) found secreted in
the serum. AFP is found in patients with hepatocellular carcinoma whereas CEA is found in
colon cancer. These are important in diagnosis.
Virus-induced tumors:
Viruses that cause tumors include
DNA viruses:
1. Papova (papilloma, polyoma) viruses. Ex. Papilloma virus causes cervical cancer.
2. Hepatitis virus: Hepatitis B virus causes hepatocellular cancer.
3. Adenoviruses
RNA viruses:
Retroviruses: Human T-lymphotropic viruses (HTLV-I and HTLV-II) causes Adult T cell
leukemia.
Virus-induced tumors express tumor-associated viral Ags (TAVA). These are cell surface
antigens that are distinct from antigens on the virion itself. However, these transplantationassociated viral Ags are shared by all tumors induced by the same virus, regardless of tissue
origin of the tumor or animal in which the tumor exists.
Chemically-induced tumors
Chemically-induced tumors are different from virally-induced tumors in that they are extremely
heterogeneous in their antigenic characteristics. Thus, any two tumors induced by the same
chemical, even in the same animal, rarely share common tumor specific antigens. These unique
antigens on chemically-induced tumors are referred to as tumor- specific transplantation antigens
(TSTA).
Syngeneic, Allogeneic and Xenogeneic Tumors:
A tumor that grows in an animal strain will also grow in another animal belonging to the same
inbred strain obtained by repeated brother-sister matings. These animals express the same MHC
molecules and are referred to as syngeneic. However, most normal animal populations are
allogeneic and have various MHC haplotypes. Thus, a tumor transferred from one animal to
another animal belonging to an outbred strain is rejected because of the allo-MHC rather than the
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TSTA.
A tumor transferred from an animal belonging to one species to another animal
belonging to a different species is rapidly rejected because the animals are xenogeneic.
Immune re
response to tu
tumors:
mors:
Evidence for immunity against malignancy comes mostly from experimental studies, wherein mice
were immunized by administering irradiated tumor cells or following removal of a primary tumor
challenged with the same live tumor. These animals were found to be resistant to rechallenge with
the same live tumor. While Abs may develop against few cancers, cell-mediated immunity plays a
critical role in tumor rejection. Thus, immunity can be transferred, in most cases, from an animal,
in which a tumor has regressed, to a nave syngeneic recipient by administration of T
lymphocytes. The T helper (Th) cells recognize the tumor Ags that may be shed from tumors and
internalized, processed and presented in association with class II MHC on antigen presenting cells.
These Th cells when activated will produce cytokines. Thus, the Th cells provide help to B cells
in Ab production. The cytokines such as IFN- may also activate macrophages to become
tumoricidal. Furthermore, the Th cells also provide help to tumor-specific cytotoxic T cells (CTL)
by inducing their proliferation and differentiation. The CTL recognize tumor Ags in the context
of class I MHC and mediate tumor cell lysis. In tumors that exhibit decreased MHC Ags, natural
killer (NK) cells are important in mediating tumor rejection.
How tumors evade immune syste
system:
According to the Immune Surveillance Theory, cancer cells that arise in the body are eliminated
by the immune system. However, due to impaired immune reactivity, the cancer cells escape
destruction.
Tumors evade immune recognition by several mechanisms. Some tumors may evade the
immune system by secreting immunosuppressive molecules such as interleukin-10 (IL-10) or
transforming growth factor-beta (TGF-) and others may induce regulatory cells particularly
the CD4+CD25+ FoxP3+ T regulatory cells or myeloid derived suppressor cells (MDSC)
which have both granulocyte and macrophage markers (Gr- 1+CD11b+). Also, some tumors
may shed their antigens which in turn may interact and block antibodies and T cells from
reacting with the tumor cells. Tumors may not express neo-antigens that are immunogenic or
they may fail to express co-stimulatory molecules required for the activation of T cells. In
addition, certain tumors are known to lack or be poor expressers of MHC antigen. Such
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tumors are however, susceptible to NK cell cytotoxicity. Another reason for failure of
immune surveillance may be the fact that in the early development of a tumor, the amount of
antigen may be too small to stimulate the immune system (low dose tolerance) or due to the
rapid proliferation of malignant cells (high dose tolerance), the immune system is quickly
overwhelmed. Tumor cells may express the death inducing ligand, FasL (CD95L) whereas the
T cells express the death receptor, Fas (CD95), thereby leading to killing of the T cells.
However, CTL have been shown to express FasL and some tumors may express Fas.
Immunothe
Immunotherapy
Immunotherapy has been used as a novel mode to treat cancer. Both active and passive means of
stimulating the non-specific and specific immune system have been employed, in some cases with
significant success.
1) Active Immunotherapy:
Wherein the host actively participates in mounting an
immune response a) Specific activation using vaccines:

i) Hepatitis B vaccine useful against development of hepatocellular cancer.
ii)
Human Papilloma virus (HPV) vaccine (Gardasil) has been successfully used to prevent
cervical cancers
b) Nonspecific activation which results in stimulation of generalized immune response is
achieved by immunization with:
i) Bacillus Calmette-Guerin (BCG) mycobacteria. ii) Corynebacterium parvum
These microbes lead to activation of macrophages which are tumoricidal.
2)
Passive Immunotherapy: This involves transfer of preformed Abs, immune cells and other
factors into the hosts.
a) Specific: Preformed Abs or CTL directed against tumor Ags are used in the treatment of
tumors i) Antibodies against tumor Ags (e.g. Abs against Her2/Neu for treatment of
breast cancer)
ii) Abs against interleukin-2 receptor (IL-2R) are used in the treatment of Human T lymphotropic
virus (HTLV-1)-induced adult T cell leukemia as this virus infects T cells and leads to production
of IL-2 that binds to IL-2R and induces the T cell proliferation.
iii) Abs against CD20 expressed on all B cells are used in the treatment of non Hodgkins
B cell lymphoma.
These Abs bind to tumor Ags on the cell surface and activate complement (C) to
mediate tumor cell lysis. In addition, Fc receptor bearing cells such as NK cells,
macrophages and granulocytes may bind to the Ag-Ab complexes on tumor cell surface
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and mediate tumor cell killing through Ab-dependent cell-mediated cytotoxicity.

iv) Abs conjugated to toxins, radioisotopes and anti-cancer drugs have also been used. These enter
the cells and inhibit protein synthesis because of the toxin. e.g. anti-CD20 conjugated to
Pseudomonas toxin or ricin toxin has been used in the treatment of B cell tumors.
There are several problems with the use of Abs
(1) Abs are not efficient because the tumor Ags are associated with class I MHC Ags.
(2) The tumors may shed Ag or Ag-Ab complexes. Thus, immune cells cannot mediate tumor
destruction.
(3) Some Abs may not be cytotoxic.
(4) Abs may bind nonspecifically to immune cells expressing the Fc receptors which include NK
cells, B cells, macrophages and granulocytes without binding to tumor cells.
v) Dendritic cells pulsed with tumor Ags may be administered which can present tumor Ags in the
context of class II MHC to tumor-specific Th cells. As tumor Ags are usually not known,
tumor lysates are used. The Th cells may in turn produce cytokines which lead to development
of CTL activity. On the other hand, the dendritic cells may be transfected with the gene for
tumor Ags, in which case, the Ags will associate with the Class I MHC and elicit a CTL
response.
b) Nonspecific:
i) Adoptive Transfer of lymphocytes:
(1) Lymphokine-activated killer (LAK) cells which are IL-2 activated T cells and NK cells can be
used in the treatment of melanoma and renal cell carcinoma
(2) Tumor-infiltrating lymphocytes (TIL) include T cells and NK cells. While the infiltrating NK
cells will kill tumors nonspecifically, the CTL will be able to kill specific tumor targets.
ii) Cytokines
(1) Interleukin-2 (IL-2): Activates T cells/NK cells which express IL-2 receptors and leads to their
proliferation. Used in the treatment of renal cell carcinoma and melanoma,
(2) Interferon-alfa (IFN): Activates NK cell activity against tumors and also used in the treatment
of Kaposi sarcoma, renal cell carcinoma and melanomas.
(3) IFN-: ncreases class II MHC expression; used in the treatment of ovarian
cancers. (4) Tumor necrosis factor (TNF)-: Kills tumor cells.
(5) Granulocyte-macrophage colony stimulating factor (GM-CSF):
neutropenia due to chemo- or radiation therapy
Useful in overcoming
iii) Cytokine gene transfected tumor cells may also be used which can activate T or LAK cells that
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can mediate anti-tumor immunity.

19. Immunodeficiency
Immunodeficiency is the failure of the immune system to protect against disease or malignancy.
Primary Immunodeficiency is caused by genetic or developmental defect in the immune system.
These defects are present at birth but may show up later on in life. Secondary or Acquired
Immunodeficiency is the loss of immune function as a result of exposure to disease agents,
environmental factors, immunosuppression, or aging.
Types of Primary Immunodeficiency Disorders
Defect in the hematopoietic stem cells results in reticular dysgenesis that leads to generalized
immune defects and subsequent susceptibility to infections. This condition is fatal if left untreated,
but can be successfully treated with bone marrow transplantation.
Myeloid Lineage deficiency: This deficiency involves myeloid progenitor cells and affects innate
immunity. Inasmuch as, phagocytosis is affected, the patients are susceptible to bacterial infections.
Congenital Agranulomatosis:
Patients have a decrease in the neutrophil count. It is due to a defect in the myeloid progenitor cell
differentiation into neutrophils. These patients are treated with granulocyte-macrophage colony
stimulating factor (GM-CSF) or G-CSF.
Chronic Granulomatous Disease (CGD):
This is characterized by defective reactive oxygen species (ROS) production which normally kills
phagocytosed bacteria. However, they exhibit inflammatory reaction with neutrophils,
macrophages and T cells resulting in the formation of granulomas. The disease is detected by a
negative reaction in the nitroblue tetrazolium test which in normal individual turns blue due to
reduction by superoxide anions. This is an autosomal recessive or X-linked trait. Treatment is
with interferon- (IFN-).
Leukocyte adhesion Deficiency (LAD):
Lack of CD18 ( chain) on T cells and macrophages impairs adhesion of these cells to
endothelium thereby preventing inflammation. Treatment is with bone marrow (devoid of T cells
and MHC-matched) transplantation or gene therapy.
Lymphoid lineage immunodeficiency:
If the lymphoid progenitor cells are defective, then both the T and B cell lineages are affected and
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result in the severe combined immunodeficiency (SCID). They are less common but are very
severe. Such infants suffer from recurrent infections especially by opportunistic microorganisms.
These include the following disorders.
Patients having both T and B cell deficiency lack recombinase activating genes (RAG1 and 2)
that are responsible for the T cell receptor and Ig gene rearrangements. These patients are
athymic and are diagnosed by examining the T cell receptor (TCR) gene rearrangement. They
also lack B cells although they do have Abs in early infant life because of passive transfer from
mother. NK cells are normal in these patients. This is an autosomal recessive trait.
Interleukin-2 Receptor (IL-2R) may be lacking in patients thereby preventing signaling by IL-2
and other cytokines which act as growth factors. This would lead to defect in the proliferation
of T cells, B cells and NK cells. This is an autosomal recessive trait.
Adenosine deaminase (ADA) is an enzyme responsible for converting adenosine to inosine.
ADA deficiency leads to accumulation of adenosine which results in production of toxic
metabolites that interfere with DNA synthesis. The patients have defects in T, B and NK cells.
These SCID are autosomal recessive traits. Treatment is by gene therapy or stem cell
transplantation.
T cell deficiency affects both cell-mediated and humoral immunity. The patients are susceptible to
viral, protozoal and fungal infections. Infection with viruses such as cytomegalovirus or attenuated
measles vaccine can be life-threatening in these patients.
DiGeorge Syndrome or congenital thymic aplasia patients lack a thymus. This deficiency results
from deletion of a region on chromosome 22 during development of 3rd and 4th pharyngeal
pouch. Because of this, this deficiency is also responsible for facial and heart defects. This is an
autosomal dominant trait. Treatment is with a thymic graft.
B cell deficiency may result from the absence of B cells, plasma cells, total Igs or selective
Igs. These patients have recurring infection with extracellular bacteria, but are capable of
eliciting an immune response against intracellular bacteria as well as viruses and fungi.
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X-linked Agammaglobulinemia (X-LA): In such patients, mature B cells are absent as they
exist in the pre B cell stage with H chains rearranged but not L chains. They also have no Igs
and suffer from recurrent bacterial infections.
X-linked hyper-IgM Syndrome: Such patients exhibit deficiency in IgG, IgA and IgE but
elevated IgM levels. This is due to a defect in the differentiation of IgM producing cells to cells
producing other Igs.
Selective Deficiency of Ig classes: IgA deficiency results from a defect in the class switching.
Such patients suffer from respiratory and genitourinary tract infections.
Common Variable Immunodeficiency: Also known as Late Onset hypogammaglobulinemia.
B cells fail to differentiate into plasma cells. Treatment is with Igs.
Complement defects:
Defects in C components results in immunodeficiency. The patients are susceptible to bacterial
infections.
Acquired or Secondary Immunodeficiencies:
All acquired immunodeficiencies have been outdone by the AIDS that is caused by Human
Immunodeficiency Virus (HIV)-1. It was first discovered in 1981 and the patients exhibited fungal
infections with opportunistic organisms such as Pneumocystis carinii and in other cases, with a skin
tumor known as Kaposi sarcoma. HIV-1 and 2 have been discovered with the strain frequently
found in N. America being HIV-1. HIV is spread through homosexual and promiscuous
heterosexual intercourse, infected blood and body fluids as well as during delivery from mother to
offspring. HIV was discovered by Luc Montagnier in Paris and Robert Gallo in Bethesda in 1983.
It is a retrovirus with RNA that is reverse transcribed to DNA by reverse transciptase (RT)
following entry into the cell. The DNA is integrated into the cell genome as a provirus that is
replicated along with the cell. HIV-1 does not replicate in most other animals but infects
chimpanzees although it does not induce AIDS in them. Severe combined immunodeficient mice
(SCID) reconstituted with human lymphocytes can be infected with HIV-1.
HIV-1 virion has an envelope made up of the outer lipid bilayer of the host cell in which are
embedded glycoproteins composed of the transmembrane gp41 along with the associated gp120.
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The gp120 binds the CD4 expressed on host cells. Within the viral envelope is the viral core or
nucleocapsid consisting of a layer of matrix protein composed of p17 and an inner capsid made up
of p24. The viral genome consists of 2 ssRNA associated with 2 reverse transcriptase (RT)
molecules as well as other enzymes including a protease and an integrase.
Replication cycle and targets of therapy:
The virus attaches to the CD4 molecule on Th cells, monocytes and dendritic cells through the
gp120 of HIV. For HIV infection, a coreceptor is required. The coreceptor is a chemokine
receptor such as CXCR4 or CCR5. CCR5 expressed predominantly on macrophages and CXCR4
on CD4+ T cells serve as coreceptors for HIV infection. After the fusion of HIV envelope and the
host membrane, the nucleocapsid enters the cell. The RT synthesizes viral DNA which is
transported to the nucleus where it integrates with the cell DNA in the form of a provirus. The
provirus can remain latent till the cell is activated when the provirus also undergoes transcription.
The virions consisting of the transcribed viral RNA and proteins are produced. These are budded
out of the host cell membrane from where they acquire the envelope. Thus, therapeutic agents have
been developed that target viral entry and fusion, as well as serve as RT, protease and integrase
inhibitors. Highly active anti-retroviral therapy is a cocktail of 3 or more such agents.
Immunological Changes:
The virus replicates rapidly and within ~2 weeks the patient develops fever. The viral load in the
blood increases significantly and peaks in 2 months after which there is a sudden decline because
of the latent virus found in germinal centers of the lymph nodes. CTL develop very early whereas
antibodies can be detected between 3-8weeks. The CTL killing of Th cells around 4-8 weeks
leads to decrease in CD4+ T cells. When the CD4+ T cell count decreases below 200/mm3, full
blown AIDS develops.
Immunotherrap
apyy:
Immunothe
There are several barriers to development of an effective HIV vaccine.
1) Attenuated vaccine itself may induce the disease
2) Heat-killed virus is not antigenic
3) CD4+ T cells may be destroyed by the vaccine
4) Antigenic variation of HIV due to induction of mutations results in escape from CTL.
5) Low immunogenicity of the virus by downregulation of MHC molecules
6) Lack of animal models in which HIV grows
7) Lack of in vitro tests to study HIV infection and growth.
The following immunological agents have been considered in developing vaccines
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1) The use of soluble CD4 which could compete for binding with gp120.
2) Anti-gp120 Abs may bind to CD4 and block entry of the HIV
3) Chemokines that compete for the co-receptors may inhibit binding and entry of HIV.
4) Immunization with deletion mutants to reduce pathogenicity.
5) Vaccination with some recombinant proteins
6) Gene encoding proteins introduced into virus vectors may be used for vaccination
7) IL-2 to boost the Th cells.
20. Antibody and Antigen Reaction
In Vitro Antigen Antibody Reactions and Their Role in the Diagnosis of Disease
There are different types of antigen antibody interaction these include
1. Precipitation reaction
2. Agglutination reaction
3. Complement fixation reaction
4. Enzyme Immuno Assay (EIA)
5. Radio Immuno Assay (RIA)
Precipitation Reaction
Precipitins can be produced against most proteins and some carbohydrates and carbohydrates- lipid
complexes. Various system are available in which precipitation tests are performed in semisolid
media such as agar or agarose, or nongel support medium such as cellulose acetate. Agar has been
found to interfere with the migration of charged particles and has been largely replaced as an
immunodiffusion medium by agarose. Agarose is a transparent, colorless, neutral gel. In the
clinical laboratory several applications of the precipitation reaction are used. These methods
include:
Immunodiffusion
Electroimmunodiffusion
Immunodiffusion: These are of two types: single and double immuodiffusion.
I. Double diffusion
This technique also referred to as the Ouchterlony method, may be used to determine the
relationship between antigen and antibodies.
Principle: Antibody dilutions and specific soluble antigens are placed in adjacent wells. If the well
size and shape, distance between wells, temperature, and incubation time are optimal, these
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solutions diffuse out, bind to each other, cross-link, and form a visible precipitate at the point of
equivalence perpendicular to the axis line between the wells the precipitation bands will be
compared with a standard antigen. The precise location of the band depends on the concentration
and rate of diffusion of antigen and antibody. In a condition of antibody excess, the band will be
located nearer the antigen well. If two antigens are present in the solution that can be recognized
by the antibody, two precipitin bands form independently. Antibodies associated with autoimmune
disorders such as rheumatoid arthritis and systemic lupus erythematosus can be identified by
double diffusion.
Identity
An identity reaction is indicated when the precipitin band forms a single smooth area. This
precipitin is formed between the antibody and the two test antigens fuses, indicating that the
antibody is precipitating identical antigen specificities in each preparation. This does not mean that
the antigens are necessarily identical; they are only identical insofar as the antibody can distinguish
the difference.
Nonidentity
A non-identity pattern (Fig 6-1B) is expressed when the precipitation line cross each other. They
intersect or cross because the sample contains no antigenic determinants in common.
Partial Identity
In a partial identity pattern, the precipitation lines merge with spur formation. This merger
indicated that the antigen are non identical but possess common determinants.
Single Radial Immunodiffusion

This is a simple and specific method for identification and quantization of a number of proteins
found in human serum and other body fluids.
Principle: Radial immunodiffusion is based on a technique using a precipitin reaction in which
specific antibody is added to a buffered agarose medium, serum containing the test antigen is
placed in a well centered in the agarose. The diameter of the resulting precipitin zone is related to
the concentration of antigen placed in a well.
Eletroimmuno diffusion (EID)
(EID
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EID is a variation of the double immunodiffusion reaction in a support medium such as cellulose
acetate or agarose through the use of an electric current that enhances the mobility of reactants and
increase their movement towards each other. Antibody is placed in the well favoring its migration
in the direction of the cathode; antigens that tend to be more negatively charged and placed in the
well that favors migration of the anode. Precipitin bands form at a point of equivalence in a shorter
periods of time. Electro immunodiffusion method, like immunodiffusion procedures, are classified
into one-or-two-dimensional, singles, or double diffusion when a voltage is applied across the gels
to move the antigens and antibodies together, immuno-double diffusion becomes counter current
immuno electrophoresis (CIE) radial immunodiffusion (RID) becomes electro immunoassay (EIA).
Counter Current immunoelectrophoresis (CIE) is a variation of the classic precipitin procedure; it
merely adds an electrical current to help antigens and antibodies move towards each other more
quickly than in simple diffusion. The procedure takes advantage of the net electric charge of the
antigens and antibodies being tested in a particular test buffer. Variables such as types of gel,
amount of current, a concentration of antigen and antibody must be carefully controlled for
maximum reactivity. The sensitivity of CIE is 10 to 20 times greater than in immuno-double
diffusion, however, it is more expensive than other techniques such as immunodiffusion.
Electro immunoassay
Antigens may be quantitiated by electrophoresis than in an antibody-containing gel electro
immunoassay. This technique combines the speed of electrophoresis with the accuracy and
sensitivity of radioimmunoassay.
Agglutination Reaction
Precipitation and agglutination are the visible expression of the aggregation of antigens and
antibodies through the formation of a framework in which antigen particles or molecules alternate
with antibody molecules. Agglutination of particles to which soluble antigen has been absorbed
produces a serum method of demonstrating precipitins. Example of artificial carriers includes latex
particles and colloidal charcoal. Cells unrelated to the antigen, such as erythrocytes coated with
antigen in a constant amount can be used as a biologic carriers. Whole bacterial cells can contain
an antigen that will bind with antibodies produced in response to that antigen when it was
introduced into the host. Agglutination tests are easy to perform and in some cases are the most
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sensitive tests currently available. These tests have a wide range of applications in the clinical
diagnosis of noninfectious immune disorders and infectious diseases.
Latex agglutination
In latex agglutination procedures, antibody molecules can be bound to the surface of latex beads.
Many antibody molecules can be bound to each latex particle, increasing the potential number of
exposed antigen-binding sites. If an antigen is present in a test specimen, the antigen will bind to
the combining sites of the antibody exposed on the surface of the latex heads, forming visible
cross-linked aggregates of latex beads and antigen. In some test systems, latex particles can be
coated with antigen. In the presence of serum antibodies, these particles agglutinate in to large
visible clumps. Examples of tests based on latex agglutination reaction include C-reactive protein,
IgG rheumatoid factors, and IgM rheumatoid factors.
Figure 92:
92: Latex agglutination reaction
Direct bacterial agglutination
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Direct whole pathogens can be used to detect antibodies directed against pathogens. The most basic
tests are those that measure the antibody produced by the host to determinants on the surface of a
bacterial agent in response to infection with that bacterium. In a thick suspension of the bacteria,
the binding of specific antibodies to surface antigens of the bacteria causes the bacteria to clump
together in visible aggregates. This type agglutination is called bacterial agglutination. Because
tube testing allows more time for antigen-antibody reaction, it is considered to be more sensitive
than slide testing.
Indirect or passive hemagglutination
Hemagglutination is agglutination of red blood cells, and tests for antibody detection. In the
indirect or passive hemagglutination technique, erythrocytes are coated with substances such as
extracts of bacterial cells, protozoa or purified polysaccharides or proteins. Erythrocyte of animals
such as sheep or rabbits, or from group O humans, function as carrier for detecting and
titrating the corresponding antibodies by agglutination. This technique is called indirect or passive
hemagglutination testing because it is not the antigen of the erythrocytes themselves but the
passively attached antigens that are bound by antibody .For example, in rubella antibody test;
erythrocytes are coated with rubella antigen. In the presence of antibody, agglutination occurs.
Hemagglutination Inhibition technique
Hemagglutination inhibition test is used to detect some viral antibodies, for example, rubella. A
known quantity of rubella viral antigen is mixed with dilutions of the patients serum, to which
red blood cells are added. If the serum lucks antibody, the virus will spontaneously attach to the
red cells, link together, and agglutinate. If antibody to the virus is present, all of the virus particles
will be bound by antibody, which prevents or inhibits hemagglutination. The serum is therefore
positive for the antibodies. The highest dilution of serum that totally inhibits agglutination of red
cells determines the antibody titer of the serum. Disadvantage of this technique include: time
consuming, & subjective bias in the interpretation for results. Negative results do not always
indicate the absence of antibody. In some case false negative results can occur from a low titer of
antibody. Nonspecific inhibitors can cause false positive results.
Complement Fixation Reaction
Complement fixation is a classic method for demonstrating the presence of antibody in serum. This
method consists of two components. The first component is an indicator system consisting of a
combination of sheep red cells; complement-fixing antibody produced against the sheep red cells in
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another animal, and an exogenous source of complement, usually guinea pig serum. When these
three components are combined in an optimal concentration, the anti-sheep cell antibody,
hemolysin, can bind to the surface of the red cells. Complement can subsequently bind to this
antigen antibody complex and cause cell lysis. The second component consists of a known antigen
and patient serum, which are added to a suspension of sheep erythrocytes, hemolysin, and a
complement. The two components of the complement fixation procedure are tested in sequence.
Patient serum is first added to the known antigen, and complement is added to the solution. If the
serum contains antibody to the antigen, the resulting antigen antibody complexes will bind all of
the complement. Sheep red cells and hemolysin are then added. If complement has not been bound
by an antigen antibody complex formed from the patient serum and known antigen, it is available
to bind to the indicator system of indicates both a lack of antibody and a negative complement
fixation test. If the patients serum does contain a complement fixing antibody appositive result
will be demonstrated by the lack of haemolysis.
Immunofluorescent Test (IFT)
The fluorescent techniques are extremely specific and sensitive. This technique consists of labeling
antibody with fluorescein isothiocyantate, a fluorescent compound with an affinity for proteins to
form a complex, conjugate. The fluorescent assay includes: direct immunofluoresent assay and
indirect immunofluoresent assay.
Direct Immunofluorescent assay
In this technique, Fluorescein- conjugated antibody is used to detect antigen- antibody reactions.
This method can be applied to the detection of hepatitis B virus & chlamydia. A fluorescent
microscope is required to observe the production of color; fluorescein gives a yellow- green light.
Indirect Immunofluorescent
Immunofluorescent Assay (IFA)
This method is based on the fact that antibodies not only react with homologous antigens but can
act as antigens and react with antibody. In the indirect immunofluorescent assay, the antigen source
to the specific antibody being tested is fixed to the surface of a microscopic slide. The patents
serum is diluted and placed on the slide to cover the antigen source. If antibody is present in the
serum, it will bind to its specific antigen unbound antibody is then removed by washing the slide,
finally antihuman globulin conjugated to a fluorescent substance that will fluoresce when exposed
to a fluorescent substance that will fluoresce when exposed to ultraviolet light is placed on the
slide. This conjugated marker of human antibody will bind to the antibody already bound to the
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antigen on the slide and will serve as a marker for the antibody when viewed under a fluorescent
microscope.
Enzyme Immuno Assay (EIA)
An enzyme labeled antibody or enzyme labeled antigen conjugate is used in immunologic assays
for detection of antigens or antibodies, in a patients serum e.g. HIV antibody, HIV antigen,
hepatitis A antibody. Various enzymes are employed in enzyme immunoassay. The most
commonly used enzymes are peroxidase and alkaline phosphatase. In EIA, a plastic bead or plastic
plate is coated with antigen. The antigen reacts with antibody in the patient serum. The bead or
plate is then incubated with an enzyme-labeled antibody conjugate, if antibody is present on the
bead or plate. The enzyme activity is measured spectrophotometrically after the addition of the
specific chromogneic substrate. Test result is calculated by comparing the spectrophotometer
reading of patient serum to that of a control serum.
Radio Immunoassay (RIA)
In radioimmunoassay, radioisotopes can be used to measure the concentration of antigen or
antibody in serum sample. If antibody concentration is being measured, radioactive labeled
antibody competes with patient unlabeled antibody for binding sites on a known amount of antigen.
The main advantage of the radioimmunoassay method is the extreme sensitivity and ability to
detect trace amounts. In addition, a large number of tests can be performed in a relatively short
time period. The disadvantage is the hazards and instability of isotopes.
Factors Affecting Antigen Antibody Reactions

Many factors affect the interaction between antigen and antibody; these include specificity, cross
reactivity, temperature PH, ionic strength, concentration, and intermolecular specificity.
Specificity: The ability of a particular antibody to combine with one antigen instead of another is
referred to as specificity. This property resides in the portion of the antigen binding fragment of an
immunoglobulin molecule. Antigen-antibody reactions can show a high level of specificity.
Specificity exists when the binging sites of antibodies directed against determinants of one antigen.
Cross reactivity: When some of the determinants of an antigen are shared by similar antigenic
determinants on the surface apparently unrelated molecules, a proportion of the antibodies directed
against one kind of antigen will also react with the other kind of antigen. This is called cross
reactivity. Antibodies directed against a protein in one species may also react in a detectable
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manner with the homologous protein in another species, which is another example of cross
reactivity.
Temperature: The optimum temperature needed to reach equilibrium in an antibody-antigen
reaction differs for different antibodies. IgM antibodies are cold reacting with thermal-range 4220C, and IgG antibodies are warm reacting, with an optimum temperature of reaction at 370C.
pH: Although the optimum pH for all reactions has not been determined, a pH of 7.0 is used for
routine laboratory testing.
Ionic strength: The concentration of salt in the reaction medium has an effect on antibody uptake
by the membrane bound erythrocyte antigens. Sodium and chloride ions in solution have inhibition
effect. These ions cluster around and partially neutralize the opposite charges on antigen and
antibody molecules, which hinders the association of antibody with antigen. Reducing or lowering
the ionic strength of a reaction medium such as low-ionic strength salt can enhance antibody
uptake.
Concentration: Under normal condition the concentration of antigen and antibody should be
optimal but sometime this thing fail to be happen in which excess antibody or antigen
concentration will result in false reaction, sometimes known as zonal reaction. When the
concentration of antigen is excess it is known as post zone reaction; excess antibody is referred as
prozone reaction. This phenomenon can by overcome by serial dilution until optimum amount of
antigen and antibody will present.
Bond strength and inter molecular attractive force Bonding of an antigen to an antibody takes place
because of the formation of multiple, reversible, intermolecular attraction between an antigen and
amino acids of the binding site. The bonding of antigen to antibody is exclusively non covalent.
The attractive force of non-covalent bonds is weak when compared to covalent bonds, but the
formation of multiple non-covalent bonds produces considerable total- binding energy. The
strength of a single antigen- antibody bond is termed antibody affinity.
The strongest bonding develops when antigens and antibodies are close to each other and when the
shapes of both the antigenic determinate and the antigen-binding site conform to each other. This
complementary matching is referred to as goodness of fit.
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21.
Serological Techniques
Materials Necessary for Basic Serologic Tests
As discussed in the previous chapter, wide verities of serologic techniques are available to detect
either an antibody or antigen. For the detection of this unknown substance from patients
specimen, the specimen should be collected and prepared appropriately. In addition the equipment
that is used for testing should be free from any contaminants so as to get true result. The following
are some of the equipment used in routine serology.
Glasswares
Dirty glass-wares easily affect serological test. After using all the glass wares (test tube, beaker,
pipette, etc.) they should be socked in detergent for several hours and rinsed several times in tap
water. Finally allow drying by placing in a dry oven or dust free place. Test tubes and pipettes
should not be scratched or broken, which will interfere with the reading of a test.
Types of glassware include:
Test tube
Glass slides
Serologic pipette with a size of l0ml, 5ml, 2ml&1ml.
Constant temperature device
Incubator and water bath are usually used in serologic tests. These materials are electrically
operated and have thermostat that hold the temperature within the required limits. These devices
should be checked prior to use by installing a thermometer
Rotating machines
Rotating machines are required to facilitate antigen antibody reactions. Such machine has a flat
plate, which rotate at a prescribed rate of speed. A knob located on the front part of the machine
controls the number of revolution per minute.
Collection Preparation and Preservation of Specimen for Serologic Tests
Specimens that are used for serologic test include: serum, plasma & cerebrospinal fluid. Serum or
plasma sample could be obtained from venous blood, which can be performed by the laboratory
personnel however. Cerebrospinal fluid should be collected by a physician or a trained nurse. For
serum or plasma sample, first 2-3 ml of venous blood is collected using sterile syringe and needle
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from a patient. If serum is required, allow the whole blood to clot at room temperature for at least
one hour and centrifuge the clotted blood for 10 minutes at 2000 rpm. Then transfer the serum to a
labeled tube with a pasture pipette and rubber bulb.
Plasma sample is obtained by treating fresh blood with an anticoagulant, centrifuge and separate
the supernatant. The specimen should be free from hemolysin blood. Finally, seal the specimen
containing tube; the tube should be labeled with full patients identification (Age, Sex, code no,
etc). The test should be performed within hours after sample collection, if this could not be done
preserve it at- 200c.
Shipment of Serological Specimen

Most health center and clinic laboratories often are limited in the diagnostic procedures that can be
carried out and have to ship serologic specimens to other laboratories. Before shipment the
following things should be considered.
Dont ship whole blood unless the tests to be performed require whole blood.
Do not inactivate serum or plasma before mailing.
Keep the specimen and packing container in the refrigerator until time of shipment but if shipment
requires several days, freeze the specimen.
Then ship the specimen by the fastest route.
Complement Inactivation
Complement inactivation is important because it is known to interfere with different tests. In
activation of complement can be achieved by heating the serum or plasma at 560C for 30 minutes.
If more than four hours has elapsed since inactivation, a specimen should be re-inactivated with
same temperature 10 minutes.
Serial Dilution
Dilution is the act of making a weaker solution from a stronger one. This is usually done by adding
a water or saline, which contains none of the material being diluted. Dilution is usually expressed
as one unit of the original solution to the total number of units of final solution. Serial dilution
means decreasing the volume of serum progressively by maintaining a constant volume of fluid
most commonly, serial dilutions are twofold, that is, each dilution is half as concentrated as the
preceding one. The total volume in each tube is the same.
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Determination of Endpoint and Titer

If we take the above example again, after serially diluting the patients serum, equal amount of
an antigen is added in each dilution to observe immunolgic reaction. The last tube that shows
visible immunologic reaction is known as end point of the test, the dilution of the antiserum at the
end point is known as the titer. The reciprocal of the greatest reacting dilution of the serum is
considered as the measure of titer or the concentration of the antibody. For example, it the highest
dilution of the serum that shows a visible reaction is at 1:32 dilution, the titer of the test is
expressed as 32.
Treponematoses
It is a chronic inflammatory disease, primarily it affects the skin and mucous membrane, and
during latent period other organs and tissues may be affected. Medically important species is
mainly T. palladium but there are other pathogenic treponemes like T. pertenue, T. endemicum &
T. carateum. Each of these organisms are obligate parasite of humans, morphologically identical,
cannot be cultured in vitro and have similar laboratory diagnosis and treatment.
These diseases differ in there:
Geographical distribution,
pathogenicity and
Degree of virulence.
T. pertenue
Cause a disease known as yaws. Its geographical distribution is West Africa, central Africa south
East Asia. T.pertenue is transmitted through exposed skin. Hand, face, legs and feet are parts of the
body most affected, it produce raised granular papilloma on the skin. In the later stage,
disfigurement of the infected area will be resulted.
T. endemicum
Cause endemic syphilis. It is widely distributed in sub-Sahara Africa and transmitted through
exposed skin and oral mucosa.
T.carateum
Cause a disease pinta, has geographical distribution of central and South America, transmitted
through exposed skin. The organisms produce itchy red papules on the uncovered part of the body.
In the later stage when infected area healed loss of the normal pigment will be resulted.
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Syphilis
Syphilis is caused by a spirochete bacterium, Treponema pallidum.
Morphology and Metabolism of T. Pallidum
Microscopically T. pallidum appears as fine, spiral (8 to 24 coils) organism, approximately 6-15
m long. They are not cultivatable with any consistency in artificial laboratory media out side the
host. T. pallidum are extremely susceptible to a variety of physical and chemical agents. However,
they may remain viable for up to 5 days in tissue specimens removed from diseased animals and
from frozen specimens. Syphilis is a venereal disease. It can be acquired by kissing a person with
active oral lesion. There are very few cases of transfusion-acquired syphilis. In addition, syphilis
may be transmitted transplacentally to the fetus. Spirochetes can be transmitted to the fetus during
the last trimester of pregnancy.
Stages of Syphilis
Untreated syphilis is a chronic disease with sub acute symptomatic periods separated by
asymptomatic intervals, during which the diagnosis can be made serologically. The progression of
untreated syphilis is generally divided in to stages. Initially, T.pallidum penetrates intact mucous
membranes or enters the body through tiny defects in the epithelium. Upon entrance, the
microorganism is carried by the circulatory system to every organ of the body. Spirochetemia
occurs very early in infection, even before the first lesions have appeared or blood tests become
reactive. Before chemical or serologic manifestations develop patients are said to be incubating
syphilis. The incubation period usually lasts about 3 weeks but can range from 10-90 days.
Primary syphilis
At the end of the incubation period, a patient develops a characteristics primary inflammatory
lesion called a chance at the point of initial inoculation and multiplication of the spirochetes. The
chancre begins as a papule and erodes to form a gradually enlarging ulcer with a clean base and
indurate edge. Generally it is relatively painless and commonly located around the genitalia, but in
about 10% of cases lesions may appear almost any where else on the body e.g. Throat, lip, hands.
Most of the patients with primary syphilis will develop swelling of inguinal lymph nods. The
primary chancre will persist for 1 to 5 weeks and will heal completely within about 4 to 6 weeks.
Primary syphilis is diagnosed by its characteristics chancre with positive serological test and
detection of T.pallidum by dark field examination from the lesion.
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Secondary syphilis
Within 2 to 8 weeks after the appearance of the primary chancre, a patient may develop the sign
and symptoms of secondary syphilis when organisms gain access to the circulation from the
infected site. The secondary stage is characterized by a generalized illness that usually begins with
symptoms suggesting a viral infection headache, sore throat, low-grade fever and occasionally
nasal discharges. Blood tests reveal a moderate increase in leukocytes with a relative increase in
lymphocytes. The disease progresses with the development of lymhadenopathy and lesions of the
skin and mucous membranes corresponding to the spread of the organism in the body by way of
the circulating blood. The lesions contain a large number of spirochetes, and when located on
exposed surfaces, are highly contagious. Macular lesions are common, and a rash invariably
involves the genitalia and often is prominent on the palms and soles. Secondary syphilis usually
resolves itself within 2 to 6 weeks, even in the absence of therapy. It may be diagnosed by typical
skin rash and positive syphilis serology test.
Latent syphilis
After resolution of untreated secondary syphilis, the patient enters a latent non-infectious state in
which diagnosis can be made only by serologic method. During the first 2-4 years of infection, one
fourth of patients will show relapses of manifestation of secondary syphilis. During these relapses,
patients are infectious, and the underlying spirochetemia may be passed translucently to the fetus.
Relapses are extremely rare after four years of latency. About one third of patients entering latency
are eventually spontaneously cured of the disease, one third will never develop further clinical
manifestation of the disease and the remaining one third will eventually develop late syphilis.
Late (Tertiary) syphilis
The first manifestations of late syphilis are usually seen from 3-10 years after primary infection.
About 15% of untreated syphilitic individuals eventually develop late benign syphilis characterized
by the presence of destructive granulomas. These granulomas, or gummas, may produce lesions
resembling segments of circles that often heal with superficial scarring. Treponems are rarely
found in the lesions, which are referred to as benign gummas. Of untreated patients 10% develop
cardiovascular manifestations. T. pallidum may damage large blood vessels such as aorta and
coronary arthritis. This condition is usually fatal. In about 8% of untreated patients, late syphilis
involves the CNS. Initially CNS disease is asymptomatic and can be detected only by examination
of cerebrospinal fluid. In symptomatic neurosyphilis, spirochetes may also involve the brain tissue
and cause destructions of the brain parenchyma (paresis), dorsal root of the spinal cord (tabes).
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Congenital syphilis
Congenital syphilis is caused by maternal spirochetemia and transplacental transmission of the
microorganism usually after 18 weeks of gestation. Congenital syphilis is diagnosed in three
fourths of the cases in patients over 10 years of age. About half of damage to the fetus depends on
the stage of the disease and the number of treponemes circulating in pregnant women at the time of
transmission. Early congenital syphilis appears either at birth or up to two years of age, the
manifestation includes cutanuous lesion, mucous membrane lesion like thick nasal discharge
containing T.pallidum. Late congenital syphilis may be characterized by fissuring around the
mouth, anus, skeletal lesions, and perforation of the palate and the collapse of nasal bones to
produce a saddle-nose deformity.
Antibodies in Syphilis
In the treponemes, two classes of antigen have been recognized those restricted to one or a few
species and those shared by many different spirochetes. Infection with T.pallidum, T.pertenue,
T.carateum T.endemicum produces similar antibody response. Specific and non-specific antibodies
are produced in the immunocompetent host.
Specific antibody
Antibodies in early or untreated early latent syphilis are predominantly IgM antibodies. The early
immune response to infection is rapidly followed by the appearance of IgG antibodies. The greatest
elevation in IgG concentration is seen in secondary syphilis.
Non specific (nontreponemal or reagin) antibodies
Are produced by infected patients against components of their own or other mammalian cells.
Reagin is widespread in nature and can be isolated from any mammalian tissues as well as from
treponemes. Although patients with syphilis almost always produce these antibodies, patients with
other infectious disease, like measles, hepatitis, leprosy, Brucelosis, malaria, rickettsia, also
produce them. Patients can alsoexhibit reagin with non-infectious conditions such as drug addition,
old age, pregnancy and recent immunization.
Collection and Handling of Syphilitic Specimen from Lesion
The treponemal lesion is infectious. As a result wearing a rubber glove is vital to protect one self
from infection. The following procedure should be followed to get a representative sample.
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Cleanse the area of chancre with swab moistened with physiological saline. Apply gentle pressure
on the area to squeeze the sample from the depth of the lesion. Collect the sample of serous
exudates on a cover glass and invert it on a slide. Deliver immediately the preparation to the
laboratory for examination by dark-field microscopy.
Tests for Syphilis
Either demonstration of microorganism in a lesion or serologic testing confirms the clinical
diagnosis of syphilis in the laboratory. The serologic methods for syphilis measure the presence of
two types of antibodies: treponemal and non treponemal.
Serologic procedures for syphilis include the following.
1. Nontreponemal method e.g. Venereal Disease research laboratory (VDRL) and the rapid plasma
regain (RPR) procedures.
2. Treponemal methods e.g. Fluorescent Treponema pallidum antibody absorption (FTA-ABS) and
microhemagglutination Treponema pallidum MHA-TP).
Nontreponemal methods
The VDRL and RPR are the two most widely used nontreponemal serologic procedures. Each is a
flocculation or agglutination test in which soluble antigen particles coalesce to form larger particles
visible as clumps when they are aggregated by antibodies. The VDRL procedure is recommended
when a patient suspected of having syphilis has a negative dark field microscopy result or when
atypical lesions are present. It is further recommended that a quantitative VDRL assessment be
made quarterly for 1 year after treatment for syphilis, or that the adequacy of treatment in both
early and latent syphilis be monitored. The VDRL procedure can be performed on cerebrospinal
fluid for the detection of neurosyphilis. The RPR test can be performed on unheated serum or
plasma using a modified VDRL antigen suspension of choline chloride with EDTA. The RPR test
card test antigen also contains charcoal for macroscopic reading. It is about as
specific as, and possibly more sensitive than, the VDRL slide test.
Treponemal methods
The FTA ABS and MHA represent treponemal methods. Reactive (Positive) regain test can be
confirmed with these two specific treponemal antigen tests. These procedures, however, should not
be used as primary screening methods. Procedures such as the FTA-ABS and MHA can be used to
confirm that a positive non-treponemal test result has been caused by syphilis rather than one of
the other biologic conditions that can produce positive VDRL, or they can determine quantitative
titer of antibody, which is useful in following response to therapy. The FTA-Abs uses a killed
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suspension of T. pallidum spirochetes as the antigen. The micro hemagglutination assay for T.
pallidum is based on agglutination by specific antibodies in the patients serum with sheep
erythrocytes sensitized to T. pallidum antigen. The Treponema pallidum immobilization test (TPI)
method is obsolete.
Sensitivity of commonly used serologic tests for syphilis

Detection of syphilis by serologic methods is related both to the stage of the disease and to the test
method. In the primary stage, about 30% of cases become serologically active after one week and
90% of patients demonstrate reactivity after three weeks. Reagin titers increase rapidly during the
first four weeks of infection and then remain stationary for approximately six months. Patients in
the secondary stage of syphilis are serologically positive. During latent syphilis there is a gradual
return of non-reactive serologic manifestations with non-treponemal method. About one third of
patients in the latent stage will remain seroreactive and presumably infectious. In late syphilis,
treponemal tests are generally reactive, non-treponemal methods are non reactive.
Venereal Disease Research Laboratory (VDRL) Qualitative Slide Test

Principle: heat inactivated serum is mixed with a buffered saline suspension of cardiolipin
lecithincholesterol antigen. This serum-antigen mixture is microscopically examined for
flocculation.
Specimen collection and preparation
The specimen should include all identification, it must include the patients full name, the date
the specimen is collected and the patients hospital identification number. Blood should be drawn
by an aseptic technique. The required specimen is a minimum of 2 ml of clotted blood. The
specimen should be promptly centrifuged and an aliquot of the serum removed. Severely lipemic or
hemolyzed serum is unsuitable for testing. Before testing, the serum must be heat in activated at
C
56 0 for 30 minutes. In activated serum should be reheated at 56 0 for 10 minutes if tested more
than 4hours after the original in activation. Cerebrospinal fluid is also an appropriate fluid for
testing.
Reagents required
VDRL antigen a colorless, alcoholic solution containing 0.03% cardiolipin, 0.9% cholesterol, and
sufficient purified lecithin to produce standard reactivity. Each lot must be serologically
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standardized by comparison with an antigen of know reactivity. Ampules should be stored in the
dark at either at 6C0 to 10 C0or at room temperature antigen that contains precipitate should be
discarded. VDRL- buffered saline Contains 1% sodium chloride, PH 6.0 + 0.1, it should be stored
in screw capped or glass stopper bottles.
Equipment required
VDRL test slide with paraffin of ceramic ring.
18 gauges hypodermic needle without bevel (it will deliver 60 drops) ml of reagents.
30 ml flat-bottomed glass with stopper, narrow mouth bottle
Syringe (1-2ml)
Rotator
Serological graduated pipette
Water bath 56C0
Preparation of working antigen suspension
1. Dispense 0.4 ml of buffered saline to the bottom of the 30ml, round, glass-stopper bottle.
2. Rapidly add 0.5ml of antigen drop by drop directly by rotating the bottle in a circular motion on
a flat surface. The pipette tip should remain in upper third of the bottle. Take care to avoid
splashing saline on the pipette. Blow the last drop of antigen from the pipette without touching the
pipette to the saline.
3. Continue to rotate the bottle for 10 seconds.
4. Add 4.1 ml of buffered saline with a 5 ml pipette
5. Place the stopper on the bottle and shake up and down approximately 30 times in 10 seconds.
The antigen suspension is ready for use, but it must be gently mixed at the time of use. Do not
force back and forth through the needle & syringe as this may lead to break down of antigen
particles and loss of their activity.
Note: The working antigen suspension can be stabilized by adding 50 l of benzoic acid to 5ml of
the diluted working solution instead of discarding within 24 hours. The temperature of the buffer
saline and antigen should be in the range of 230 to 29C0. The antigen suspension must be used on
the day of preparation.
Quality control
Include positive control sera of graded reactivity each time serologic testing is performed. The
antigen suspension to be used each day is first examined with these control sera. Store control sera
frozen at -200C or liquid form for 7 to 10 days. Thaw, mix thoroughly, and heat in activate at
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560C before use. Check antigen dispensing needle at the time of use to be sure that it accurately
deliver 60-drops/ml reagents. Clean needles and syringes by rinsing with water, alcohol and
acetone. Remove needle from syringe after cleaning.
Procedure
1. Pipette 0.5 ml of inactivated patient serum in to one of the rings of the ceramic-ringed slide.
Pipette additional specimen and controls in to additional rings.
2. Add one drop of antigen suspension to each serum with a calibrated 18-gauge needle and
syringe held in a vertical position.
3. Rotate the slide on a mechanical rotator for 4 minutes. In externally dry climate, cover the slide
with a lid containing moistened filter paper to prevent evaporation during rotation.
4. Examine each specimen microscopically with the low (10x) objective.
Note: The test should be performed at a temperature range of 230-290C.
Reporting Results
Non reactive: No clumping or very slight roughness weakly reactive: Small clumps
Reactive: medium and large clumps
Weakly reactive: small clumps
Note: All reactive and weakly reactive specimens (sera) should be tested quantitatively to estimate
the antibody titer.
False negative reactions it can occur in a variety of situations like:
Technical error (e.g. unsatisfactory antigen preparation or techniques.
The presence of inhibitors in the patients serum
Low antibody titer patients may have syphilis, but the reagin concentration is too low to produce a
reactive test result.
It may be caused by several factors: an infection that is too recent to have produced antibodies, the
effect of treatment, latent or inactive disease, or patients who have not produced protective
antibodies because of immunological tolerance. These seronegative patients may demonstrate a
positive reaction with more sensitive treponemal tests such as the FTA-ABS.
Inappropriate temperature
Prozone reaction
Weakly reactive results can be caused by
Very early infection
Lessening of the activity of the disease after treatment.
Improper technique or questionable reagents
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False positive reactions can also be observed. Of all positive serologic tests for syphilis, 10% to
30% may be false biologic positive reactions. Non-syphilitic positive VDRL reactions have been
reported with cardiolipin type of antigen in rheumatic fever, pneumococcal pneumonia, infectious
hepatitis, leprosy, malaria, pregnancy, aging individuals, and rheumatoid arthritis. Contaminated or
hemolyzed specimens can also produce false positive results.
VDRL quantitative test
Principle: Retest quantitatively to an end-point titer all sera that produce reactive, weakly reactive
or questionably nonreactive results in the qualitative VDRL slide test.
Same as VDRL Qualitative test for undiluted serum
Preparation of serial dilution.
A. Pipette 0.05 ml of 0.9% saline in to ring number 2,3 &4 on ceramic slide do not spread the saline.
B. Pipette 0.05 ml of serum to ring numbers 1 and 2. Draw the serum and saline mixture up and down
in the pipette tip in ring number 2 to mix. Aspirate 0.05 ml of diluted serum and spread the
remaining dilution over the entire area of the circle with the pipette tip.
C. Transfer 0.05ml of the diluted (1:2) serum in ring number 2 to ring number 3. Draw the serum and
saline mixture. Aspirate 0.05 ml of diluted serum and spread the remaining dilution over the entire
area of the circle with the pipette tip.
D. Transfer 0.05 ml of the diluted (1:4) serum in ring number 3 to ring number 4 Draw the serum and
saline mixture up and down in the pipette tip in ring number 3 to mix. Aspirate 0.05 ml of diluted
serum and spread the remaining dilution over the entire area of the circle with the pipette tip.
Discard 0.05 ml of the diluted (1:8) serum from ring number 4 unless greater dilutions are needed
for strongly relative serum, and spread the remaining dilution over the entire area of the circle with
the pipette tip.
Reagent, Supplies, and equipment
In addition to the VDRL qualitative test the following reagent and piece of equipment are needed.
0.9% saline
Preparation weigh 0.9gm of sodium chloride to a leit volumetric flask. Dilute to the calibration
mark with distilled water
Safety pipette (50ml or 0.05ml)
Procedure:
1. Add one drop of antigen suspension to each diluted serum with a calibrated 18-gauge needle and
syringe held in a vertical position.
2. Rotate the slide on a mechanical rotator for 4 minutes.
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3. Examine each specimen microscopically (10x objective)

0
Note: the test should be performed at a temperature range of 23 to 29 C.

Reporting results
Report the titer in terms of the highest dilution that produces a reactive (weakly reactive) result.
Rapid Plasma Reagin (RPR) Card Test
Principle
A cardiolopin lecithin-cholesterol antigen coated with carbon particle is mixed with patients
serum. Then flocculation reaction is observed macroscopically in the presence of cardiolipin
antigen.
No special preparation is required before specimen collection. The specimen should be labeled
with all patients identification fresh serum or plasma sample can be used. It is not important to
heat inactivate the specimen before testing.
Reagents & Equipment
Note: Except for the antigen, all other components should be stored at room temperature in a dry
place in the original kit packaging.
Provided in the test kit
RPR card test antigen
It contains cardiolipin, lecithin, cholesterol, EDTA, charcoal, chorine chloride, and distilled water.
Store the antigen suspension in ampules or in plastic dispensing bottle at 20 to 80C. Unopened
ampules have a shelf life of 12 months from the date of manufacture.
Opened antigen ampules has stability for 3 months
Needle, 18-gauge, without bevel. The needle should deliver 60+2 drops of antigen suspension per
milliliter when held in a vertical position.
Specially prepared, plastic-coated cards
Serological pipette
Dispenser, 0.05 ml/drop
Stirrer
Other material
Rotator
Humidifier cover containing a moistened sponge
0.9% saline
Fluorescent Treponema Pallidum Antibody
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Absorption Test
Principle: Patients serum is added on the slide coated with T. pallidum antigen followed by
addition of fluorescent-tagged anti-human globulin and examine under fluorescent microscope.
Its the most sensitive confirmatory test. Specimen collection and preparation. The serum should
be heat inactivated. The specimen should be labeled with all the identification.
Reagent supplied
Treponema pallidum antigen: Extracted from rabbit testicular tissue
Store at 60
c -100C in lyophilized form
Conjugate (fluorescent labeled anti-human globulin)
Positive control
Equipment
. Test tubes, pipette
. Incubator
. Fluorescent microscope
Procedure
1. Coat the slide with T.pallidum antigen by adding a drop of suspension of T.pallidum on a clean
slide and keep in oven at low temperature
2. Take out and wash by rinsing with tap water to remove excess unbounded T.pallidum
3. Add the patients serum to the coated antigen and incubate, rinse by tap water to remove
excess antibody, if the pt serum has an antibody, and to remove the whole serum if it doesnt
contain antibody.
4. Add conjugate (fluorescent tagged antihuman globulin) and rinse with tap water, to remove
excess conjugate, if serum contain antibody and to remove the whole conjugate, if it doesnt
contain antibody.
5. Examine under fluorescent microscope.
The fluorochromes usually used are
Fluorescein isothyocyanate yellow green
Rudamin - Red color
Reporting results
Fluorescence indicates the presence of specific antibody to T.pallidum. Non fluorescence indicate
the absence of specific antibody to T.pallidum.
Preparation of Control Sera
1. Collect all reactive sera and store in freezer
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2. Collect all non reactive sera and store in freezer

3. After collecting a large amount thaw at room temperature
4. Filter to remove the particles
5. Add mertiolet (preservative) 1 mg/ml serum.
6. Make a serial dilution of reactive with non reactive sera
7. Perform several tests each day. Select the dilution, which always gives reactive, weakly reactive
and non-reactive result.
8. Prepare a large quantity of those dilutions
9. Distribute in a small container (alginate) and store in a freezer.
10. Control sera of graded reactivity should be included each time when serologic procedures are
performed.
NB: If the control sera fail to give the desired (known) result (do not produce the established
relativity) pattern the result of the specimen is unacceptable so to have acceptable result the
following should be done:
- Prepare another antigen suspension
- Test temperature must be adjusted at room temperature (23-290C)
- Adjust equipment
- Use commercially prepared controls
- Strictly follow the manufactures procedure.
Agglutination Test for Febrile Disease
When any pathogenic microorganism invades the human body, the natural response is the
production of antibodies. The host and microbial factors influence the rate of antibody formation,
the type and amount of antibodies produced, and the persistence of antibody in the circulation.
Among the antibodies produced in response to certain pathogenic microorganism are febrile
agglutinins. The microorganism that elicits the production of febrile agglutinin is characterized by
presence of persistent fever & frequently difficult to grow in laboratory cultures. Some of the
causative agents of febrile diseases are salmonella species, rickettsial and brucella abortus.
Typhoid and Paratyphoid Fever
The etiological agent is Salmonella species; it occurs in human only. Sometimes it is termed as
enteric fever since they colonize the intestine. Salmonella of medically important species are
S.typhi (typhoid fever), S.paratyphi A and B (paratyphoid fever). Typhoid and paratyphoid fever is
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transmitted through ingestion of contaminated food or water. They contaminate usually by carriers
like rodents, hens, cows, etc. Typhoid or enteric fever is a clinical syndrome characterized by
fever, headache, splenomegaly, leucopenia & cough. Its incubation period ranges from 7 to 14
days. In 5% to10% of untreated patients relapse may occur the symptoms in relapse are milder than
the initial illness and begin about two weeks after discontinuation of antimicrobial therapy. The
carrier state is assymptomatic and in 1 - 3% of carriers there is continuous excretion of S.typhi for
a minimum of one year. The gall bladder is the site of persistent intestinal infection.
Identification of salmonella
Salmonella species can be identified based on their antigenic structure they possess. They have
three different antigenic structures.
O- antigen (somatic antigen)
It is lipopolysaccharide of the outer membrane, which is heat and alcohol stable antigen.
Salmonella is divided in to five distinct serogroups (A-E) on the basis of somatic antigen.
H-antigen (flagellar antigen)
H-antigen is protein, which makes the perithrchous flagella. It is heat and alcohol labile.
Salmonella is further subdivided in to more than 1200 serotypes on the basis of flagellar antigens.
Vi- Antigen:
This is the antigen that determines the virulence, the ability to cause disease, of the organism.
Preparation of antigen suspension
Salmonella antigen suspension is available commercially and its also possible to prepare in the
laboratory.
A. Preparation of H antigen (flagellar antigen)
Procedure
1. Inoculate bacteria from a single colony in to a broth and incubate for 6hrs.
2. View a drop of the culture in a wet film to confirm that most of the bacteria are motile and
therefore sufficiently flagellated for the tests.
3. Kill the culture by adding formaldehyde to a concentration of 0.2% and incubate for several
hours at 370C
B. Preparation of O antigen
Procedure
1. Suspend the bacteria from an agar culture in saline and heat for 30 minute at 1000C to remove
the flagella.
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2. Centrifuge to separate the bacteria from the detached flagella.

3. Resuspend the bacteria in saline.
Alternatively
1. Remove the flagella by mixing a dense saline suspension of the bacteria with an equal volume of
absolute ethanol
2. Incubate for 20 hr at 370C
3. Dilute the suspension with saline.
Widal test
Widal test is a serological test, which is commonly used to diagnose typhoid and paratyphoid
fever. The patients serum is tested for O and H antibodies
Rapid slide (Screening) test
1. Clean the glass slides supplied in the kit well and wipe it free of water.
2. Place one drop of undiluted test serum in each of the first circle (1to4) and one drop of positive
control serum in each of the last two circles.
3. Place one drop of antigen O, H, A (H) and B (H) in circle 1,2,3, &4 respectively and O antigen
in circle five and H antigen in circle 6
4. Mix the contents of each circle with separate applicator stick and spread to fill the whole area of
the individual circle.
5. Rotate the slide for one minute and observe for agglutination.
If agglutination is visible, quantitative estimation of the titer of the appropriate antibodies should
be done
Tube agglutination method
Procedure
1. Take a set of 8 clean dry test tubes for each serum to be tested.
2. Place 1.9ml of saline in tube 1 and 1 ml of saline in other tuber (2-8)
3. Transfer 0.1 ml of undiluted serum to tube 1. Mix thoroughly. The resultant dilution of serum is
1:20.
4. Further dilutions are done in the following
a) Transfer 1ml of the diluted serum from tube 1 and place in tube 2 this leads to 1:40 dilutions in
tube 2
b) Repeat the transfer process for tube 7 after mixing.
c) Leave 1 ml of saline in tube 8 at the saline control
Note. Tube 1 has a serum dilution of 1:20, 1:40 (2), 1:80 (3), and 1:160 (4), 1:320 (5), 1:640 (6) &
1:1280 (7).
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5. Add one drop of appropriate antigen in each (use only that antigen suspension which has given a
positive reaction in the screening test).
Note: Each antigen (O, H, AH, BH) will require a series of 8 tubes for determine the titer of their
corresponding antibodies.
6. Mix well and incubate overnight (16-18 hrs) at 370C.
7. Examine agglutination macroscopically.
8. Two tubes for positive control O and it antigen should be included.
Interpretation
1. Only a titer above 1:80 should be considered as significant.
2. A rise in titer (done each week) is considered to be definite evidence of infection. A single test
result is considered of diagnostic value only when it is usually high (above 160).
3. Antibiotic treatment in typhoid fever often prevents a rise in titer,
4. A negative test does not rule out the possibility of infection because of the tine when the blood
sample was taken in relation to the stage of the disease.
5. Positive results should always be interpreted with reference to clinical findings.
Rickettsial Disease
Rickettsiae resemble viruses in that they are obligate intercellular parasite and unable to survive as
free-living organisms. They are about the size of the large viruses and can just be seen with the
light microscope. Unlike viruses rickettsiae contain both RNA and DNA multiply by binary
fission, they have cell wall that contains muramic acid and enzyme. Based on their antigenic
structure, the genus rickettsia has been divided into three main groups: typhus group (R.
prowazeki, R. typhi), scrub typhus group (R. tsutsugamushi), and spotted fever group (R.conori,
R.siberica, and R.rickettsi R.conoripijperi). Man is an accidental host of rickettsia species except R.
prowazeki; they live in intestinal tract of louse, fleas, ticks and mites. Reservoirs host include,
dogs, rats, mice, rodents Rickettsial disease can be acquired by inhaling of dried infected vector
faces, through damaged skin bite of an infected vector ticks, mite, etc. The infection is
characterized by high continuous fever, severe head ach and body pains, marked weakness,
enlarged spleen
Laboratory diagnosis
Embryonated egg inoculation technique used for culturing viruses can also be used for isolating
rickettsiae however it require costly materials and performed in reference laboratory.
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Serology
In rickettsial infection, specific IgM antibodies are produced followed by IgG response in the later
stages. The most reliable and useful serological techniques to diagnose rickettsial infections are
immunofluorescent assay and complement fixation test; however, this test is not available in
district laboratory due to its cost.
WeilWeil-felix reaction
A Weil Felix test is a type of agglutination test most commonly used serologic test. The reaction is
based on similarity of particular antigenic determinant, which occur in most species of pathogenic
rickettsia and in the OX-19, OX -2 strains of Proteus vulgaris and OX-K strains of Proteus
mirabilis. In other word, Proteus antigen is used to detect rickettsial antibody. This could be an
example of hetrophile antigen antibody reaction. Weil Felix test has similar principle and
procedure with Widal test.
Note: False negative reactions are common in scrub typhus. False positive reactions may occur in
Proteus infections, relapsing fever, brucellosis and other acute febrile illnesses. A rise in titer in
two consecutive specimen collected in interval is significant than a rise in titer in single specimen
and a rise in titer in single specimen should not be taken as a positive sample.
Brucella Abortus
Brucella aborts, gram-negative bacilli, is the causative microorganism of brucellosis. If is a
zoonoses that infects humans by accident. The agents of brucellosis are normal flora of the genital
and urinary tract of many animals including pigs, cows, and dogs. Most human acquire brucellosis
because of the ingestion of contaminated food products or through occupational exposure. Farmers,
veterinarians, and slaughterhouse workers are particularly prone to infection.
Because of the difficulty of isolating this organism by the culturing technique, many cases of
brucellosis are diagnosed serologically by identifying the presence of antibodies. Antibodies
usually appear within 2 to 3 weeks after infection. An antibody titer of 1:80 to 1:60 strongly
suggests infection.
Human Chorionic Gonadotropin Hormon (HCG)
HCG and Pregnancy
Human chorionic gonadotropin (HCG) is a hormone secreted by placenta during pregnancy. Its
production stimulates secretion of progesterone by the ovary. Adequate levels of progesterone are
necessary for successful implantation and prevent any further release of egg from the ovary.
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Human chorionic gonadotrophin is a glycoprotein, has alpha and beta sub units. The alpha subunit
usually cross reacts with the alpha subunit of leutenizing hormone; however, the beta subunit is
specific for HCG. It appears in urine, blood and amniotic fluid. The serum and urine level rise
rapidly during gestation, reaching a peak at six to eight weeks, after which there is a steady
decline.
Pregnancy Tests
Laboratory tests for pregnancy are based on the detection of human chorionic gonadotrophin
hormone in serum or urine; mainly there are two types of test.
I. Biologic animal Bioassay (A-Z test)
This test is performed in laboratory animal (female mouse). i.e. Patients urine is injected in to a
female mouse after certain period, the mouse will be killed and the ovary will be examined for sign
of pregnancy. However, this test cannot be used for early diagnosis. Moreover it is time consuming
and requires steady supply of laboratory animals.
II.. Immunologic test
Immunologic test could be qualitative and quantitative. Qualitative estimation of HCG in urine is
used for early detection and confirmation of pregnancy. Quantitative estimation of HCG in serum
has of value in case of preeclamptic toxemia, hydatidiform mole and choriocarcinoma. Compared
to biologic animal assay, immunologic test is less expensive and quicker test.
10.3 Specimen Collection
An early morning urine specimen is preferable because this is the most concentrated and contains
the highest level of HCG. However, specimen collected at any time may be used with a specific
gravity at least 1.010. Urine must be collected in a clean detergent free container. If it cannot be
tested immediately, it should be refrigerated at 40C for not longer than 48 hours. Specimen
preserved with boric acid is also suitable for testing. When tested, the urine and test reagents
should be at room temperature. If the urine is cloudy it should be filtered or centrifuged and the
supernatant fluid used. Specimens that are heavily contaminated or contains large amount of
proteins or blood, are not usually suitable for testing.
There are two types of immunologic test commonly available and provided in a form of kit.
Rapid latex slide test: have two types
I. Indirect latex slide test
II. Direct latex slide test
Tube test (haemagglutination inhibition technique)
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A. Rapid latex slide test

I. Indirect latex slide test
Principle
Urine specimen is first treated with anti-HCG and then reacted with the latex suspension. If the
urine contains HCG, the anti HCG will be neutralized and then the latter will not be available to
the HCG coated latex particles for bringing about agglutination.
Reagents and materials
Antiserum that contain HCG antibody
Latex reagent coated with HCG
Positive and negative controls
Mixing sticks and slides
Procedure
Note: In all case it is better to refer manufacturer manual
1. Place one drop of urine sample on the ring of the slide provided by the manufacture. 2. Add one
drop of anti-HCG reagent to the urine specimen placed on the slide. Mix the two fluids well with
applicator stick.
Note. In order to maintain the same volume, always hold the dropper in the same vertical position
and use the same vertical position and use the same kind of dropper for both urine specimen and
the antiserum.
3. Rock the slide gently for about 30 seconds
4. Gently shake the vial with latex antigen and then add one drop.
5. Mix again with applicator stick and observe the appearance of agglutination at 2 minutes under a
bright light.
Reporting
Latex particle agglutinated _______ Negative (non-reactive)
Homogenous suspension of latex particles without any sign of agglutination _______Positive
(Reactive)
II. Direct latex slide test
Principle
The reaction is based on the reaction between HCG in urine and the latex particles coated with anti
HCG. In positive result agglutination will be observed.
B. Haemagglutination inhibition test (tube test)
It is more sensitive than slide test
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Principle: similar with latex slide test except the HCG is coated on red cells, not on polystyrene
particles.
Procedure
1. Add a drop of urine and drop of anti-HCG antiserum in a small tube.
2. Add red cell coated with HCG
3. Mix the contents of the tube and leave at room temperature (20-280C) for 1-2 hrs to allow time
for the red cells to settle.
4. If the urine contains HCG it will combine with the antibody. This will leave no antibody to react
with the HCG on the red cells.
5. If the urine contains no HCG, the anti HCG antibody will react with the HCG on the red cells
and cause their agglutination.
Reporting
Reactive __________Non-agglutinated cells settle in the bottom of the tube.
Non-reactive _______Agglutinated cells settle and cover the bottom of the tube.
Factors Affecting Pregnancy Tests
Different factors influence the result of pregnancy test these are False Negative may occur in
conditions like:
Error in reading inappropriate interpretation of procedure
Test is performed too early-The concentration of HCG is below the sensitivity of the test, which is
capable of detecting reliably. The sensitivity of a test, the recommended time of testing will be
included in the information supplied by the manufacture.
Urine too diluted -falsely low levels of HCG may be due to a diluted urine (low specific gravity)
Ectopic pregnancy implantation of the ovum outside the uterine cavity
False positive may occur in conditions like
Error in reading- inappropriate interpretation of test procedure
Luteinizing hormone cross-reaction
Test performed at time of ovulation or in menopausal women
Proteinuria and hematuria
Recent pregnancy -test performed less than 10 days after abortion of full-term delivery.
Detergents on glassware and slide used in the test, it must be well rinsed to remove trace of
detergent even the smallest trace of detergents may affect the performance of the test.
Drug interference- aldomet, marijuana, aspirin in large doses, etc.
HCG treatment for infertility
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Trophoblastic disease e.g. molar pregnancy or choriocarcinoma

HCG secreted by malignant tumor (ovary, breast, lung, kidney)
Testicular tumor (in male)
Use of pregnancy test
Situations in which pregnancy testing is indicated include pregnancy test usually ordered to
investigate some conditions like ectopic pregnancy, threatened abortion, hydatiform mole, and
choriocarcinoma. It also used for checking a woman of childbearing age is pregnant before
carrying out medical or surgical investigation, x-ray or drug therapy that could be harmful to an
embryo.
22. Human Immunodeficiency Virus (HIV)
Disease Characteristics and Clinical Manifestation
Infection with HIV produces a chronic infection with symptoms that range from a symptomatic to
the end stage complications of AIDS. Typically, patients in the early stages of HIV infection are
either completely asymptomatic or may show mild, chronic swelling of lymph nodes. The early
phase may last from many months to many years after viral exposure. During the early period after
primary infection, widespread dissemination of virus occurs and a sharp decrease in the number of
CD4 T- Cells in peripheral blood is manifested. The early burst of virus in the blood is often
accompanied by flulike symptom. This phase is followed by a prolonged period of clinical latency
range 7 to 11 years. During this period the patient is usually a symptomatic. Due to different
factors, there is a variation in the duration of clinical latency. The quantity of CD4 lymphocytes
continues to diminish as the disease progresses and when the number of cells reaches a critically
low level the risk of opportunistic infection increases. Clinical symptoms of the later phase of the
disease include extreme weight loss, fever and multiple secondary infections. The end stage of
AIDS is characterized by the occurrence of opportunistic infection like M. tuberculosis,
Salmonella, P. carinii, etc.
Laboratory Diagnosis
A window period of seronegativity exists from the time of initial infection to 6 or 12 weeks or
longer. Serological screening tests designed to detect HIV antibodies are usually enzyme linked
immunosorbent assay and dot blot assay; western blot assay is commonly used confirmatory test.
Enzyme Linked ImmunoSorbent Assay (ELISA)
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The indirect ELISA is the most commonly utilized test that is supplied in the form of kit. In this
type of assay, an antigen coated on a solid phase combine with the patients serum containing
antibody, the antigen antibody complex will interact with conjugate (enzyme Labeled with anti
human immunoglobulin) then a color change is observed up on addition of a substrate. The
intensity of the color gives an indication of the amount of bound antibody.
Dot blot assay (HIV spot test)
Dot blot assay are rapid and easy to perform. In this type of assay antigens are coated on micro
particles that are trapped within a membrane. These antigen bind with HIV antibody on the
patients sample than a color production is observed when the conjugate is added on antigen
antibody complex.
Western blot assay
Western blot assay is the most widely accepted confirmatory assay for the detection of HIV;
however, it is time consuming and expensive test. In the western blot procedure, purified HIV-1
viral antigens are electrophoresed on sulfate polyacryamide gel (SDS gels) and the separated
polypeptides are then transferred on to sheets of nitrocellulose paper incubated with the serum
specimen. Any antibody that binds to the separated peptides present on the nitrocellulose paper is
detected by a secondary antihuman antibody, conjugated to enzyme substrate. Antibody
specificities against known viral components are considered true positive results, whereas
antibodies specific against non-viral cellular contaminants are nonspecific, false-positive results.
Viral hepatitis is the most common liver disease worldwide. The viral agents of acute hepatitis can
be divided in to twomajor groups
1. 10 hepatitis viruses: A, B, C, D & E
2. 20 hepatitis viruses: Epstein- Barr virus, cytomegalovirus, herpes virus, etc.
Primary hepatitis viruses attack primarily the liver and have little direct effect on other organ
system. Secondary viruses involve the liver secondary in the curse of systemic infection of another
body system.
Hepatitis A virus (Infectious or shortshort- incubation hepatitis)
Hepatitis A virus is a small, single stranded RNA virus when seen by electron microscope.
Infection can be acquired by ingestion of virus in contaminated food or water from hands or other
objects contaminated with infected feces (fecal routes), after exposure within 2-6 weeks clinical
symptoms will develop. In acute phase of infection, the highest titers of HAV can be detected in
stool sample. Shortly after the onset of fecal shedding, an IgM antibody is detectable in serum,
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followed within a few days by the appearance of an IgG antibody. IgM anti-HA is almost always
detectable in patients with acute HA. IgG anti-HA, a manifestation of immunity, peaks after the
acute illness and remains detectable indefinitely, perhaps lifelong. The finding of IgM anti- HA in
a patient with acute viral hepatitis is highly diagnostic of acute HA. Demonstration of IgG anti-HA
indicates previous infection. The presence of IgG anti-HA protects against subsequent infection
with HA virus, but it is not protective against HBV or other viruses.
Testing methods for hepatitis A virus include the following:
1. Total antibody by enzyme immunoassay (EIA)
2. IgM antibody by RIA
3. HA antigen by radioimmunoassayt (RIA)
Hepatitis B virus
(Long term or serum hepatitis)
Hepatitis B virus is a double stranded, DNA virus. It is the classic example of a virus acquired
through blood transfusion. It has various antigens hepatitis B surface antigen, an outer coat,
hepatitis B antigen that is an inner core component and hepatitis B core antigen. The major routes
of transmission of hepatitis B virus included blood transfusion, sexual inter course, transplacental
and sharing of contaminated needle. The incubation period of hepatitis B virus may range from 626 weeks. Infected patients manifest hepatitis B virus in all body fluids including blood, feces,
urine, saliva, semen, tear and milk.
Serum that is collected in acute stage of illness can be tested by:
Counterimmunoelectrophoresis
Enzyme Linked immuno sorbent assay
Reverse passive Hemagglutination test
Reverse passive hemagglutination is the commonly employed test since it is less expensive and
sensitive test.
Hepatitis C virus
Viral hepatitis caused by hepatitis c virus, the identification of this virus is not clear, sometime it
known as non-A/non-B hepatitis. This virus is commonly acquired by contaminated blood and
blood products.
Hepatitis C virus antibody can be detected from serum usually by radioimmunoassay.
Page 171
Hepatitis D virus (Delta virus)

It is defective or incomplete RNA virus that is unable by itself to cause infection, i.e., transmitted
through blood products. HBV is required as a helper to initiate delta infection only persons with
acute or chronic HBV infection can be infected with delta agent.
Laboratory diagnosisdiagnosis- Radio immuno assay
Hepatitis E virus
This is responsible for large water borne out breaks incubation period 6 weeks.
Laboratory diagnosis: Specific test for IgM & IgG antihepatitis E virus.
C - reactive protein
The main biologic sign of inflammation is an increase in the erythrocyte sedimentation rate (ESR).
In addition an increase in plasma concentrations of a group of proteins known as acute-phase
proteins is a good indicator of local inflammatory activities and tissue damage. The acute phase
proteins include C-reactive protein (CRP), inflammatory mediators (e.g. complement components
c3 and c4, fibrinogen, etc. CRP is prominent among the acute-phase proteins because it provides
fast and adequate information of the actual clinical situation; as a result CRP is a direct and
quantitative measure of the acute-phase reactions.
Measures of CRP add to the diagnostic procedure in selected cases (e-g. in the differentiation
between a bacterial and a viral infection). An extremely elevated CRP is suggestive of a possible
bacterial infection. The CRP level may be useful also for monitoring the effect of treatment and for
early detection of postoperative complications or intercurrent infections. The CRP is a parameter
for inflammatory activity. CRP is a method of choice for screening for inflammatory and malignant
diseases and monitoring therapy in inflammatory disease. Elevations of CRP occur in nearly to
diseases states, including bacterial infection, viral infections, and myocardial infarction specificity
rules out CRP as a definitive diagnostic tool.
The CRP test has been widely used to detect infection in circumstances where microbial diagnosis
is difficult. These conditions include septicemia and meningitis in neonates, infections in
immunosuppressed patients, serious post operative infections etc. CRP levels rise following the
tissue injury or surgery. In uncomplicated cases the level of CRP peaks about 2 days
postoperatively and gradually returns to normal levels within 7 to 10 days. CRP is synthesized
more rapidly than other acute phase proteins; assays of CRP are the measurement of choice in
suspected inflammatory conditions.
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Tests for CRP

Rapid latex agglutination test
Principle: The test is based on the reaction between patient serum containing CRP as the antigen &
the corresponding antibody coated to the treated surface of latex particle. The coated particles
enhance the detection of an agglutinate reaction when antigen is present in the serum being tested.
Specimen- Serum
Reagent & materials required
CRP latex reagent
Glycine saline buffer
Capillary pipette
Applicator sticks
Glass slide
Serologic pipettes & rubber bulb
Quality control
Include positive & negative control serum.
Procedure
1. Deliver one drop of undiluted serum on a slide by using capillary Pipette.
2. Deliver one drop of positive and negative control on separate (other) circle of the slide
3. Add one drop of CRP latex reagent to each serum specimen & to each control
4. Mix the suspension using separate applicator sticks.
5. Tilt the slide back & forth slowly for two minutes observe for agglutination macroscopically.
Reporting
Positive reaction agglutination
Negative reaction absence of agglutination
Streptolysin O
Streptolysin O is a hemolytic factor produced by most strains of GroupA beta- hemolytic
streptococci (S. pyogenes). Streptococci are gram-positive cocci in chain, non-motile, facultative
anaerobes. It produce toxin like streptolysin O & streptolysin S and enzymes like DNAase,
streptokinase. It is oxygen & heat labile immunogenic enzyme with molecular weight range from
50,000-75,000 Dalton, which cause lysis of red cells under reduced condition. It can severely
damage or destroy PMN leukocytes, also able to destroy adjacent cells and tissues and thus
contribute to the spread of organism from local sites.
Antistreptolysin O (ASO)
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Is specific neutralizing antibody produced after infection with these organisms & it appears in
serum from 1 week-1month after the onset of a streptococcal infection. It combines and neutralizes
the heamolytic activity of streptolysin O.
Streptolysin S
Oxygen stable non-antigenic toxic enzyme with molecular weight of 20,000 Dalton. It hemolyze
red cells and phagocytic cells by direct cell to cell contact also it is responsible for the surface
heterolysis observed around colonies of group A streptococci grown on blood agar plate.
Serological
Serological test
Antistreptolysin O test is used to diagnose conditions post streptococcal resulting from a
streptococcal infection especially in diagnosis of rheumatic fever and glomerulonephritis when
its not possible to isolate Group A streptococci in culture (most complication develop at a stage
when it is not possible to isolate group A streptococcus in culture). The principle of the test
depends on the following factors. Antistretolysin O can react specifically with SLO and inhibits the
heamolytic activity. The amount of ASO can be estimated by dilution of patients serum in the
presence of constant amount of SLO to the point where there is still complete prevention of
haemolysis. The occurrence of ASO depends on the production of SLO by streptococci in the
infected host.
Commercially available test are:
Antistreptolysin O latex slide test- used for screening a significant raise in ASO titer
Antistreptolysin O titration test used to determine the titer of ASO antibody.
Rapid Antistreptolysin O latex agglutination test

Principle: In the presence of ASO antibody a visible agglutination reaction will be exhibited when
a serum specimen combine with latex particle coated with streptolysin
O antigen.
Specimen
Clear, haemolysis free serum
Reagent & equipment required
Latex particle coated with streptolycin
0.9% NaCl solution
Glass slide with six cells
Applicator sticks (stirrer)
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Control reagent
Other material required
Timer
Test tubes
Pasture pipettes and rubber bulb
Serologic pipette and safety bulb
Quality control
Positive control-a prediluted serum containing at least 200 lu/ml of ASO. This control should
exhibit visible agglutination at the end of the 3-minute test period. Negative control serum a
prediluted serum containing less than 100 Iu/ml of ASO. This control should exhibit a smooth or
slightly granular appearance at the end of the 3-minute test period. A positive and negative control
should be tested and read concurrently with each group of patient sera.
Procedure
1. Dilute the serum by saline in 1:2
2. Label the slide for positive control dilution negative control and patient sera
3. Pipette 50l of the controls and patient sera onto the appropriately labeled cell (well)
4. Add one drop of latex reagent to each well
5. Mix the specimen with separate applicator stick spread the mixture evenly over the well.
Rotate the slide for exactly 3 minutes. Examine with a bright light
Reporting
Positive agglutination
Negative- No agglutination
Agglutination demonstrates 200 lu/ml or more ASO. Positive results should be retested
quantitatively.
A titer of 200Iu/ml or greater may be associated with rheumatic fever or glomerulonephritis.
Antistreptolysin O titration kit
In the titration test, a constant amount of streptolysin O antigen reagent is added to a series of
dilutions of the patients serum. Following a period of incubation, Group O washed human or
rabbit red cells are added. The tubes are then examined for lysis of the red cells. Haemolysis
occurs in those tuber in which there is insufficient antibody to neutralize the antigen. The highest
dilution of serum showing no haemolysis is the ASO titer; the titer of ASO antibody in the serum
is directly proportional to the reciprocal of the serum dilution.
Bibliography
Page 175
1. Cheesbrough Monica. Medical Laboratory Manual for Tropical Countries; vol ll

2000.Cambridge Butter worth.Heinemann.Ltd.
2. P.Stities Daniel. Basic and Clinical Immunology; 8thed, 1994,USA 4.Fischbach Frances,
Manual of Laboratory and Diagnostic tests; 4ed 1992,Lippincott.
3. 5. Turgeon L.M, Immunology and Serology in Laboratory Medicine,2nded, 1996,Mosby.
4. Male, Brostoff, Roth and Roitt: Immunology. 6th ed.
5. Male, Brostoff, Roth and Roitt: Immunology. 7th ed.
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Desalegn Amenu (M.Sc.) Wollega University, 2014