Programme M.

Sc BT Course code: 19BTP06 Course Title: Immunotechnology

Section A (10 x 1 = 10)

1. (D) Repeated infection
2. (A) BM
3. (D) Ig G
4. (D) Ig E
5. (B ) HAT Medium
6. (A) Antigen
7. (B) Initiated by haptens
8. (C) Ig E
9. (D) Tumor suppressor genes
10. (A) Immunotherapy

Section – B (5 x5 = 25)
11 A.
The mechanism of humoral immunity is accomplished after interaction of antigen with B-cells, antibody production
and antigen-antibody binding.
1. Interaction of antigen with B-cells:
The B-cells after production from the stem cells migrate to lymphoid organs. When antigen interacts and contacts the
receptor, the later gets stimulated. The B-cells differentiate into antibody producing cells through three steps:
activation, proliferation and differentiation
Moreover, from the beginning it passes through several stages such as lymphoid stem cell, progenitor B-cell, pre-B-
cell, immature B-cell, mature B-cell, activated B-cell, plasma cell and memory cell.
The plasma cells divide to produce a large number of genetically identical cells known as clones. Some of the
progeny cells are differentiated into the antibody producing cells i.e. plasma cells. The plasma cells secrete
antibodies specific to antigens. Some of the other B-cells act as memory cells. Each plasma cell is capable to
produce about 2,000 antibodies per second but they remain alive for a few days only.

2. Production of antibodies:
Antibody production may or may not depend on T-antigens.

Antibodies production against T-dependent antigens:

Antibody production by B-cells is also cooperated by the other cells such as T-cells and macrophages. For example,
B-cells produce antibodies in cooperation with T-cells against a type of antigen known as T-dependent antigen (Fig.
22.9). The T-dependent antigen consists of bacteria, some of proteins, RBCs, etc.
The macrophages or dentric cells (highly branched cells) play a significant role. The B-cells need the help of antigen
presenting cells (APC) (i.e. the macrophages and dentric cells that attack the T-dependent antigens and partially
digest them) and specialized helper T-cells to produce antibodies against T-dependent antigens.
This process is accomplished in the following steps:
The APC attaches the T-dependent antigens and partially digests them. The APC usually takes up the polypeptide
fragments of the antigen and keep them on the cell surface. The specific T-cells which are known as helper T-cells
interact with the APC. The antigen fragments and certain self antigens are recognised by helper T-cells and carry
them on the surface of APC.
The self antigens are actually the cell surface proteins that are the components of the major histocompatibility
complex. They serve as the self antigens, the signals by which the immune system distinguishes the self from non-
self.
The foreign antigen is recognised by the helper T-cells only when it gets combined with a self antigen on the surface
of APC. This results in stimulation of suitable B-cells. For that the possession of self antigens by B-cells is very
necessary.
Helper T-cells are required by all immune responses that induce IgG, Ig and IgE. However, a single complex of the
antigen and the self antigen present on the cell surface of B-cells are recognised by T-cell receptors.

11 B.

Antigen-Presenting Cells (APCs)

All nucleated cells in the body have mechanisms for processing and presenting antigens in association with MHC
molecules. This signals the immune system, indicating whether the cell is normal and healthy or infected with an
intracellular pathogen. However, only macrophages, dendritic cells, and B cells have the ability to present antigens
specifically for the purpose of activating T cells; for this reason, these types of cells are sometimes referred to as
antigen-presenting cells (APCs).
Antigen Presentation with MHC II Molecules
MHC II molecules are only found on the surface of APCs. Macrophages and dendritic cells use similar mechanisms
for processing and presentation of antigens and their epitopes in association with MHC II; B cells use somewhat
different mechanisms that will be described further in B Lymphocytes and Humoral Immunity. For now, we will focus
on the steps of the process as they pertain to dendritic cells.
After a dendritic cell recognizes and attaches to a pathogen cell, the pathogen is internalized by phagocytosis and is
initially contained within a phagosome. Lysosomes containing antimicrobial enzymes and chemicals fuse with the
phagosome to create a phagolysosome, where degradation of the pathogen for antigen processing begins. Proteases
(protein-degrading) are especially important in antigen processing because only protein antigen epitopes are
presented to T cells by MHC II.
APCs do not present all possible epitopes to T cells; only a selection of the most antigenic or
immunodominantepitopes are presented. The mechanism by which epitopes are selected for processing and
presentation by an APC is complicated and not well understood; however, once the most antigenic, immunodominant
epitopes have been processed, they associate within the antigen-binding cleft of MHC II molecules and are
translocated to the cell surface of the dendritic cell for presentation to T cells.
Antigen Presentation with MHC I Molecules
A cell becomes infected with an intracellular pathogen (e.g., a virus), protein antigens specific to the pathogen are
processed in the proteasomes and bind with MHC I molecules for presentation on the cell surface. This presentation
of pathogen-specific antigens with MHC I signals that the infected cell must be targeted for destruction along with the
pathogen.
The MHC class I antigen presentation pathway. (1) Proteins are proteolytically processed in the cytosol by the
proteasome. (2) Peptides generated by the proteasome are translocated into the ER lumen by TAP. (3) MHC class I
molecules (heavy chain and associated β2M) fold and assemble in the ER lumen with the aid of the ER chaperones
calnexin, calreticulum and ERP57. (4) The MHC class I molecule in a complex with calreticulum and ERP57
associates with TAP and tapasin facilitates peptide binding. (5) Peptide loaded MHC class I molecules dissociate
from TAP and are transported through the secretory pathway to the plasma membrane.
The usual process of antigen presentation through the MHC I molecule is based on an interaction between the T-cell
receptor and a peptide bound to the MHC class I molecule. There is also an interaction between the CD8+ molecule
on the surface of the T cell and non-peptide binding regions on the MHC class I molecule. Thus, peptide presented in
complex with MHC class I can only be recognised by CD8+ T cells. This interaction is a part of so-called ‘three-signal
activation model’, and actually represents the first signal. The next signal is the interaction between CD80/86 on the
APC and CD28 on the surface of the T cell, followed by a third signal – the production of cytokines by the APC which
fully activates the T cell to provide a specific response.

12A.

Antigenicity is the capacity of a chemical structure (either an antigen or hapten) to bind specifically with a group of
certain products that have adaptive immunity: T cell receptors or antibodies
There are some essential factors which influence the power of antigen
Those are:
i. Molecular size,
ii. Structural stability,
iii. Degradability,
iv. Foreignness,
v. Chemical composition and heterogeneity,
vi. Antigen processing and presentation,
vii. Conformation and
viii. Accessibility.

12B

The complement system or "complements" the ability of antibodies and phagocytic cells to clear pathogens from an
organism. The complement system consists of a number of small proteins found in the blood, generally synthesized
by the liver as a part of the acute phase reaction during systemic inflammation (from TNF-alpha release). When
stimulated by one of several triggers, proteases in the system cleave specific proteins to release cytokines and
initiate an amplifying cascade of further cleavages. The end result of this activation cascade is massive amplification
of the response and activation of the cell-killing membrane attack complex (also called MAC). There are three
different pathways by which the complement system may occur.

Classical Complement Pathway

The classical complement pathway is the main pathway by which the complement system occurs. It is comprised of a
cascade of many steps with complement proteins cleaving one another in a sequential order:
The antibody binds to an antigen on the surface of a pathogen, activating the C1 complement protein.
C1 acts a protease and cleaves C2 and C4 to form C4b2b.
C42b converts C3 into C3a and C3b, which forms a C5 convertase.
C5 convertase cleaves C5 into C5a and C5b.
C5b forms a complex with C6, C7, and then C8, and C9, which becomes the membrane attack complex that lyses the
pathogen.
Note that C5a has a number of other functions in the immune system, such as causing vasodilation during
inflammation and stimulating neutrophil chemotaxis. Additionally, the body's cells express a glycoprotein called decay
accelerating factor, which decays C3 and C5 convertase on the body's cells. This factor prevents membrane attack
complexes from forming on the body's cells under normal conditions.

13A
 Immunoelectrophoresis refers to precipitation in agar under an electric field.
 It is a process of a combination of immuno-diffusion and electrophoresis.
 An antigen mixture is first separated into its component parts by electrophoresis and then tested by double
immuno-diffusion.
 Antigens are placed into wells cut in a gel (without antibody) and electrophoresed. A trough is then cut in the
gel into which antibodies are placed.
 The antibodies diffuse laterally to meet diffusing antigens, and lattice formation and precipitation occur
permitting determination of the nature of the antigens.
  The term “immunoelectrophoresis” was first coined by Grabar and Williams in 1953.

Principle of Immunoelectrophoresis
When an electric current is applied to a slide layered with gel, the antigen mixture placed in wells is separated into
individual antigen components according to their charge and size. Following electrophoresis, the separated antigens
are reacted with specific antisera placed in troughs parallel to the electrophoretic migration and diffusion is allowed to
occur. Antiserum present in the trough moves toward the antigen components resulting in the formation of separate
precipitin lines in 18-24 hrs, each indicating reaction between individual proteins with its antibody.
Procedure of Immunoelectrophoresis
1. Agarose gel is prepared on a glass slide put in a horizontal position.
2. Using the sample template, wells are borne on the application zone carefully.
3. The sample is diluted 2:3 with protein diluent solution (20μl antigen solution +10 μl diluent).
4. Using a 5 μl pipette, 5 μl of control and sample is applied across each corresponding slit (Control slit and
Sample slit).
5. The gel is placed into the electrophoresis chamber with the samples on the cathodic side, and
electrophoresis runs for 20 mins/ 100 volts.
6. After electrophoresis completes, 20 μl of the corresponding antiserum is added to troughs in a moist
chamber and incubated for 18- 20 hours at room temperature in a horizontal position.
7. The agarose gel is placed on a horizontal position and dried with blotter sheets.
8. The gel in saline solution is soaked for 10 minutes and the drying and washing repeated twice again.
9. The gel is dried at a temperature less than 70°C and may be stained with protein staining solution for about
3 minutes followed by decolorizing the gel for 5 minutes in distaining solution baths.
10. The gel is dried and results evaluated.
Results of Immunoelectrophoresis
1. The presence of elliptical precipitin arcs represents antigen-antibody interaction.
2. The absence of the formation of precipitate suggests no reaction.
3. Different antigens (proteins) can be identified based on the intensity, shape, and position of the precipitation
lines.
Applications of Immunoelectrophoresis
1. The test helps in the identification and approximate quantization of various proteins present in the serum.
Immunoelectrophoresis created a breakthrough in protein identification and in immunology.
2. Immunoelectrophoresis is used in patients with suspected monoclonal and polyclonal gammopathies.
3. The method is used to detect normal as well as abnormal proteins, such as myeloma proteins in human
serum.
4. Used to analyze complex protein mixtures containing different antigens.
5. The medical diagnostic use is of value where certain proteins are suspected of being absent (e.g.,
hypogammaglobulinemia) or overproduced (e.g., multiple myeloma).
6. This method is useful to monitor antigen and antigen-antibody purity and to identify a single antigen in a
mixture of antigens.
7. Immunoelectrophoresis is an older method for qualitative analysis of M-proteins in serum and urine.
8. Immunoelectrophoresis aids in the diagnosis and evaluation of the therapeutic response in many disease
states affecting the immune system.
Advantages of Immunoelectrophoresis
1. Immunoelectrophoresis is a powerful analytical technique with high resolving power as it combines
separation of antigens by electrophoresis with immunodiffusion against an antiserum.
2. The main advantage of immunoelectrophoresis is that a number of antigens can be identified in serum.
Limitations of Immunoelectrophoresis
1. Immunoelectrophoresis is slower, less sensitive, and more difficult to interpret than
Immunofixation electrophoresis.
2. IEP fails to detect some small monoclonal M-proteins because the most rapidly migrating immunoglobulins
present in the highest concentrations may obscure the presence of small M-proteins.
3. The use of immunoelectrophoresis in food analysis is limited by the availability of specific antibodies.

13B
Immunomodulators act at different levels of the immune system. Therefore different kinds of drugs have been
developed that selectively either inhibit or intensify the specific populations and subpopulations of immune responsive
cells, i.e. lymphocytes, macrophages, neutrophils, natural killer (NK) cells, and cytotoxic T lymphocytes (CTL).
Immunomodulators affect the cells producing soluble mediators such as cytokines (5). Thus, in immunotherapy the
immune system is targeted in order to help the healing of a given disease.
Immunosuppressants inhibit the immune response in organ transplantation and autoimmune diseases, whereas
immunostimulants increase the immune response in infections, immunodeficiency (for example AIDS) and cancers.
The term immunomodulation is used rather than immunostimulator for a substance that causes measurable
alterations in immune function. Their action can be specific or nonspecific.

Immunomodulatory drugs modify the response of the immune system by increasing (immunostimulators) or
decreasing (immunosuppressives) the production of serum antibodies . Immunostimulators are prescribed to
enhance the immune response against infectious diseases, tumours, primary or secondary immunodeficiency, and
alterations in antibody transfer, among others . Immunosuppressive drugs are used to reduce the immune response
against transplanted organs and to treat autoimmune diseases such as pemphigus, lupus, or allergies

14A
Type II Hypersensitivity:
Type II hypersensitive reactions are those in which tissue or cell damage is the direct result of the actions of antibody
and complement.
(i) Mode of action:
This type of reaction is resulted by blood- transfusion reactions in which host antibodies react with foreign antigens
present on the incompatible transfused blood cells and mediate destruction of these cells.

Antibody can mediate cell destruction by activating the complement system to create pores in the membrane of the
foreign cell by forming membrane attack complex (MAC). This can also be mediated by antibody dependent cell-
mediated cytotoxicity (ADCC).
A faulty cross-matching leads to haemolysis of the donor’s erythrocytes in the blood vessels of the recipient due to
the alloantigen of the donor’s erythrocytes react with the antibodies in the serum of the recipient and in combination
with activated complement, the erythrocytes undergo haemolysis

(ii) Biological effect:

 1. Haemolytic disease of the newborn develops when maternal IgG antibodies specific for foetal blood-group antigens
cross the placenta and destroy foetal red blood cells. Severe haemolytic disease of the new born is called
erythroblastosis foetalis, when an Rh+ foetus expresses an Rh antigen on its blood cells that the Rh – mother does not
express it

3. Certain antibiotics (e.g. penicillin, cephalosporin and streptomycin) can absorb non- specifically to proteins
on RBC membranes, forming a complex similar to a hapten-carrier complex and gradually induces anaemia
called drug-induced haemolytic anaemia.

14B

Systemic lupus erythematosus (SLE) is an autoimmune disease. In this disease, the immune system of the body
mistakenly attacks healthy tissue. It can affect the skin, joints, kidneys, brain, and other organs.
It is characterized by states of exacerbation and remission Systemic lupus erythematosus (SLE) is a multi-system
auto-immune disease that is caused by tissue damage resulting from antibody and complement fixing immune
complex deposition.
Causes
The cause of SLE is not clearly known. It may be linked to the following factors:
 1. Genetic 2. Environmental 3. Hormonal 4.Certain medicines
SLE is more common in women than men. It may occur at any age. However, it appears most often in people
between the ages of 15 and 44. The disease affects African Americans and Asians more than people from other
races.
Symptoms
Symptoms vary from person to person, and may come and go. Everyone with SLE has joint pain and swelling at
some time. Some develop arthritis. SLE often affects the joints of the fingers, hands, wrists, and knees.
Other common symptoms include:
 Chest pain when taking a deep breath. - Fatigue. - Fever with no other cause. - General discomfort, uneasiness, or ill
feeling (malaise). - Hair loss. - Weight loss. - Mouth sores. - Sensitivity to sunlight.
 Skin rash: A "butterfly" rash develops in about half the people with SLE. The rash is mostly seen over the cheeks and
bridge of the nose. It can be widespread. It gets worse in sunlight.
Other symptoms depend on which part of the body is affected:
 Brain and nervous system: Headaches, numbness, tingling, seizures, vision problems, and personality changes
 Digestive tract: Abdominal pain, nausea, and vomiting
 Heart: Valve problems, inflammation of heart muscle
 Lung: Buildup of fluid in the pleural space, difficulty breathing
 Skin: Patchy skin color and fingers that change color when cold (Raynaud phenomenon)
 Kidney: Swelling in the legs
15 A
An oncogene is a gene that has the potential to cause cancer. In tumor cells, these genes are often mutated, or
expressed at high levels. Most normal cells will undergo a programmed form of rapid cell death (apoptosis) when
critical functions are altered and malfunctioning.
There are two classes of oncogenes — one is viral oncogene present in viruses that causes the transformation of
target cells. The counterpart of which stays within the host cell involved in normal cellular functions are called cellular
oncogenes. The cellular sequences themselves are not oncogenic, are described as proto-oncogenes, whose
capture by retrovirus and subsequent modification may create an oncogene.
Oncogenic potential of tumour virus resides in a single function or a group of related functions that are active early in
the viral lytic cycle. Tumour viruses carry genes (v-one) which confer on them the ability to convert host cell into
tumourigenic state. About 100 viral oncogenes have been identified so far.
When transformation occurs, the relevant genes are integrated into the genomes of transformed cells and expressed
constitutively. Oncogenes of DNA tumour viruses do not have cellular counterparts. In case of non- defective RNA
viruses, tumourigenicity does not rely upon an individual viral oncogene, but upon the ability of the virus to activate a
cellular proto- oncogene.
Acute transforming retroviruses capture cellular oncogene (absent in ancestral virus) by means of a transduction
event during an infective cycle. At least 25 c-onc genes have been identified by their representation in retroviruses.
Viral infection is not really necessary for tumour formation as evidenced by transfection assay.
Proto-oncogenes may also be activated by insertion, translocations or amplification of DNA sequences although
nucleotide sequence remains unaltered (c-myc). Proto-oncogenes can be activated by mutation (c-ras). Activation of
‘tumour suppressor genes’ may also be responsible for unconstrained growth of cancer cells.
When we compare a viral oncogene (v-onc) sequence with a corresponding cellular oncogene (c-onc) sequence, we
find that the organization of the viral gene corresponds to the mRNA of the c-onc gene, but the v-onc genes consist of
uninterrupted coding sequences, while the c-onc genes have the usual genomic organisation of alternating exon and
intron.
The cellular oncogenes and proto-oncogenes have multiple exons separated by introns, whereas the viral oncogenes
are single exons. For example, chicken cellular src proto-oncogene contains 11 introns separating 12 coding
sequences, whereas the RSV v-src gene has a single, uninterrupted coding sequence.
Both the genes code for protein kinases that phosphorylate tyrosine residues, ^both the protein can interact with
same antibody. It has been found that there are 18 single nucleotide pair differences between the coding sequences
of v-src and c-src that result in 8 amino acid changes in the protein products, but those must not be involved in
oncogenecity.
In several cases, the v-onc coding sequence appears to have evolved only relatively little from the c-onc sequence,
usually by point mutations.
An indication that the v-onc genes retain functions related to their c-onc progenitors, is provided by the fact that the
majority of nucleotide changes are located in third base positions where they do not affect the protein sequence. So,
in a word, viral oncogenes and cellular oncogenes are almost same, but the cellular oncogenes are present as proto-
oncogene.
These proto-oncogenes can mutate to form that are capable of inducing oncogenesis.

15B.
A recombinant vaccine is a vaccine produced through recombinant DNA technology. This involves inserting the
DNA encoding an antigen (such as a bacterial surface protein) that stimulates an immune response into bacterial or
mammalian cells, expressing the antigen in these cells and then purifying it from them.
Recombinant Vaccines—General:
Recombinant DNA technology in recent years has become a boon to produce new generation vaccines. By this
approach, some of the limitations (listed above) of traditional vaccine production could be overcome. In addition,
several new strategies, involving gene manipulation are being tried to create novel recombinant vaccines.
Types of Recombinant Vaccines:
The recombinant vaccines may be broadly categorized into three groups:
1. Subunit recombinant vaccines:
These are the components of the pathogenic organisms. Subunit vaccines include proteins, peptides and DNA.
2. Attenuated recombinant vaccines:
These are the genetically modified pathogenic organisms (bacteria or viruses) that are made non-pathogenic and
used as vaccines.
3. Vector recombinant vaccines:
These are the genetically modified viral vectors that can be used as vaccines against certain pathogens. Some of the
developments made in the production of recombinant vaccines against certain diseases are briefly described.
Type # 1. Subunit Vaccines:
As already stated, subunit recombinant vaccines are the components (proteins, peptides, DNAs) of the pathogenic
organisms. The advantages of these vaccines include their purity in preparation, stability and safe use. The
disadvantages are — high cost factor and possible alteration in native conformation. Scientists carefully evaluate the
pros and cons of subunit vaccines for each disease, and proceed on the considered merits.
Hepatitis B:
Hepatitis B is a widespread disease in man. It primarily affects liver causing chronic hepatitis, cirrhosis and liver
cancer. Hepatitis B virus is a 42 nm particle, called Dane particle. It consists of a core containing a viral genome
(DNA) surrounded by a phospholipid envelope carrying surface antigens (Fig. 16.1 A).
Infection with hepatitis B virus produced Dane particles and 22 nm sized particles. The latter contain surface antigens
which are more immunogenic. It is however, very difficult to grow hepatitis B virus in mammalian cell culture and
produce surface antigens.

The gene encoding for hepatitis B surface antigen (HBsAg)

has been identified. Recombinant hepatitis B vaccine as a
subunit vaccine, is produced by cloning HbsAg gene in
yeast cells. Saccharomyces cerevisiae, a harmless baking
and brewing yeast, is used for this purpose (Fig. 16.1B).
The gene for HBsAg is inserted (pMA 56) which is linked to
the alcohol dehydrogenase promoter. These plasmids are
then transferred and cultured.
The cells grown in tryptophan, free medium are selected
and cloned. The yeast cells are cultured. The HBsAg gene
is expressed to produce 2nm sized particles similar to
those found in patients infected with hepatitis B. (These
particles are immunoreactive with anti-HBsAg antibodies).
The subunit HBsAg as 22 nm particles can be isolated and
used to immunize individuals against hepatitis B.
Hepatitis B vaccine-the first synthetic vaccine:
In 1987, the recombinant vaccine for hepatitis B (i.e.
HBsAg) became the first synthetic vaccine for public use. It
was marketed by trade names Recombivax and Engerix-B.
Hepatitis B vaccine is safe to use, very effective and
produces no allergic reactions. For these reasons, this
recombinant vaccine has been in use since 1987.
The individuals must be administered three doses over a
period of six months. Immunization against hepatitis B is
strongly recommended to anyone coming in contact with
blood or body secretions. All the health professionals—
physicians, surgeons, medical laboratory technicians,
nurses, dentists, besides police officers, firefighters etc.,
must get vaccinated against hepatitis B.

Section C (5 x 8 = 40)

16A.
Thymus:
Thymus is a greyish, flat, bilobed lymphoid organ situated above the heart and extending into the neck on the front
and sites of trachea. It develops from the epithelium of third and fourth pharyngeal pouches and, on maturity, acts as
the site of development and maturation of lymphocytes named thymus-derived lymphocytes or T-lymphocytes or T-
cells.
Each lobe of thymus is surrounded by a capsule and is divided into a series of lobules, which are separated from
each other by strands of connective tissue called trabeculae. Each lobule is organized into two compartments-outer
and inner. The outer component is called cortex, whereas the inner component is called medulla.
The cortex is densely packed with thymocytes, whereas the medulla is sparsely populated with thymocytes.
Thymocytes develop from prothymocytes. The latter are produced in bone marrow, migrate through blood stream,
enter the cortex of the thymus, and act as thymocytes. Thymocytes divide rapidly in the cortex and give rise to T-
lymphocytes.
Of the T-lymphocytes produced in thymus only 5% leave the thymus as viable cells. Though the reason for this
apparent wasteful process is not known, some believe that it is the elimination of T-lymphocyte clones that react
against self.

Both the cortex and the medulla of the thymus are criss-crossed by a three dimensional network consisting of
epithelial cells, dendritic cells, and macrophages, which make up the framework of the organ and contribute to the
growth and maturation, of thymocytes.
Some epithelial cells of the outer cortex possess long membrane extensions that surround as many as 50
thymocytes. These cells are called nurse cells. Other epithelial cells of the cortex have long interconnecting
cytoplasmic extensions that form a network and have been found to interact with many of the thymocytes when they
traverse the cortex.
The function of the thymus is to generate T-lymphocytes and to confer immunological competence on to them during
their stay in the organ. T-lymphocytes so educated in the thymus become capable of mounting cell-mediated immune
response against appropriate antigen.
This is effected under the influence of the thymic microenvironment and several hormones such as thymosin and
thymopietin produced by the epithelial cells of the thymus. The competent T-lymphocytes immediately move from
thymus to the secondary or peripheral lymphoid organs.
Lymph Nodes:
Lymph nodes are small, encapsulated, bean-shaped structures clustered at junctions of the lymphatic vessels which
are distributed throughout the body. Lymph nodes contain a reticular network packed with lymphocytes,
macrophages and dendritic cells, and filter out pathogenic microorganisms and antigens from the lymph.
As the lymph percolates through a lymph node, any pathogen or antigen that is brought in with the lymph is trapped
by the phagocytic cells and dendritic cells.
A lymph node consists of three regions: the cortex, the paracortex, and the medulla (Fig. 42.3). Cortex is the
outermost region and contains several rounded aggregates of lymphocytes (mostly B-lymphocytes), macrophages,
and follicular dendritic cells arranged in primary follicles. Each follicle has a pale-staining germinal centre surrounded
by small dark-staining lymphocytes.
The deeper region lying beneath the cortex is the paracortex. It is the zone between the cortex and the medulla.
Paracortex possesses large number of T-lymphocytes and also contains inter-digitating dendritic cells thought to
have migrated from tissues to the lymph node.
Because of the presence of large number of T-lymphocytes in it. the Para-cortex is also referred to as a thymus-
dependent area in contrast to the cortex which is a thymus-independent area. Medulla, the inner most region of
lymph node, is more sparsely populated with lymphoid-lineage cells. Of the lymphoid-lineage cells present, many are
plasma cells actively secreting antibody molecules.
Each lymph node has a number of lymph vessels called afferent lymphatic vessels, which pierce the capsule of a
lymph node at numerous sites and empty lymph into the sub-capsular sinus. The lymph now percolates slowly inward
through the cortex, paracortex, and medulla, allowing phagocytic cells and dendritic cells to trap pathogens and
antigens carried by the lymph.
The lymph then is drained into a single large lymph vessels called efferent lymphatic vessel that carries the lymph to
the thoracic duct, which empties into a large vein in the neck.

16B.
The common lymphoid progenitor cells give rise to B-lymphocytes (B-cells) that differentiate into antibody
secreting plasma cells. T-lymphocytes (T-cells) that become activated T-cells. natural killer (NK) cells, and some
dentritic cells.
The common myeloid progenitor cells give rise to erythroblasts that produce erythrocytes (red blood cells),
megakaryoblasts that produce platelets (thrombocytes), myeloblasts that produce granulocytes (eosinophils,
basophils, neutrophils), monoblasts that differentiate into monocytes which give rise to macrophages and dendritic
cells, and an unknown precursor that produces mast cells.

17A
Classically, an antigen is defined as an organism, a molecule, or part of a molecule or substance which may be self
or non-self, can evoke noticeable immune response and can bound distinctively with antibodies. Antigenicity is the
ability of antigen to combine specifically with the final products of immune system i.e., either with antibodies or with
cell surface receptors.
Antigens, which are able to induce adaptive immunity, are called immunogens. All immunogens are antigens unless
their ability to stimulate an immune response is significant.
Nature of Antigens:
Classification of Antigens:
Antigens can be classified under two major categories, called:
(1) Exogenous
(2) Endogenous antigens – i. Xeno-genic or Heterogenic antigens
ii. Allogenic or Idiotypic antigens iii. Autologous antigens.
Antigens can also be classified into two broad categories:
1. Comparison of T-independent and
2. T-dependent antigens.
Essential Features of Antigens:
There are two essential features found within the antigenic molecules:
1. The molecules must be recognized as foreign by the host.
2. After antigen processing, antigens must undergo some physical and chemical changes that can stimulate the
immune system.

17B

Antibodies are specific glycoproteins produced by В-cells and constitute main components of humoral immunity.
Antigen or immunogen present in the foreign body (microorganism or parasite) are mainly protein molecules which
are responsible for the production of specific antibody or immunoglobin in the host.
Structure of Immunoglobulins:
Generally, immunoglobulins contain one or more basic units (monomers) having four polypeptide chains united by
disulphide bonds (S – S). Immunoglobulin G or IgG is the most common and thoroughly studied antibody molecule. It
has Y – shaped structure with following domains:
1. Heavy chains:
Entire immunoglobulin is a tetramer consisting of two identical high molecular weight or heavy chains and two low
molecular weight or light chains. Heavy chain is made of about 500 amino acids, (mol. weight of 50,000 to 70,000
Daltons). There are 5 types of heavy chains designated α (alpha), δ (delta), Ɛ (epsilon) γ (gamma) and µ (mu) in IgA,
IgD, IgE, IgG and IgM respectively.
ADVERTISEMENTS:
Both chains of antibody molecule have variable (V) and a constant (C) portion. Generally, half of each light chain
consisted of a variable region (VL) whereas only one-quarter of each heavy chain was variable (VH) among different
patients. Remaining three-quarters of the heavy chain (CH) was constant for all IgGs.
The constant portion of heavy chain is subdivided into three sections of almost equal length. It is believed that each of
the three sections of С part of IgG heavy chain arose during evolution by the duplication of an ancestral gene which
encoded for an Ig unit of approximately 110 amino acids. The variable regions (VH or VL) are also thought to have
arisen by evolution from same ancestral Ig units.
2. Light chains:
They include two low molecular weight chains designated K (Kappa) and λ. (lambda) present in five different
antibodies. About 200 – 250 amino acids are present in each chain with mol. weight of 23,000 Daltons). Both chains
show following domains:
(a) Antigen-binding domain:
They consist of Fv and Fab segments. Among them, Fab segment is heterodimer being made of entire light chain +
VH and CH1 parts of heavy chains. Fv segment is also heterodimer containing variable domains of heavy chains
(VH) and light chains (VL).
(b) Effector domains:
They are also called Fc segment which helps in the elimination of antigen. Fc region consists of homodimer with
СH2 and CH3 domains of both heavy chains.
Structurally, terminal regions of both light and heavy chains contain three small hyper variable regions forming
antigen binding sites. These are also called CDR or complementarity-determining regions. These hyper variable
portions of the chains contain deletions and insertions of amino acids as well as substitutions of one amino acid for
another.
About 5-10 amino acids in such hyper variable region form the antigen binding site. Therefore, variable domains of an
antibody determine the molecule’s combining site specificity.
 Classes of Immunoglobulins:
1. IgG:
It is the major class of serum immunoglobulins. It takes part in various antibacterial, antiviral and antitoxic activities. It
passes through placenta and gives passive immunity to the new born child for about six months.
2. IgM:
It is roughly less than 10% of normal serum immunoglobulins. It is made up of five-four-chain subunits. Its molecular
weight is 900,000 Daltons. IgM develops primary immunity against most antigens. IgM antibodies are effective
against toxins from diptheria, tetanus, botulism, anthrax, snake venoms.
3. IgA:
IgA antibody is about 13 to 15% of the total Igs in human serum. It is a predominant immunoglobulin in body serum
and secretions e.g. saliva, tears, nasal fluids, sweat, colostrum, milk and lung secretions, gastrointestinal and
genitourinary secretions. Here it provides defence against microorganisms.
Amniotic fluid also has IgA and it provides passive immunity to the foetus. Serum IgA contains several types of
antibodies including isoagglutinins, antibodies of anti-diptheria, anti-Brucella, anti-insulin and antipoliomyelitis.
Secretory IgA is structurally different from serum IgA. Serum IgA does not pass from serum to exocrine glands or to
other mucosal sites. Serum IgA is produced by plasma cells in the bone marrow and Secretory IgA is produced by
local plasma cells distributed in Secretory tissues.
4. IgD:
It is found in traces in human serum, i.e. about 1% of total serum Igs. It was first discovered in the serum of patients
of myeloma tumors or malignant lymphomas. Its function is not yet established. Its structure is like that of IgG having
two L chains and two H chains joined by S-S bonds. Its molecular weight is about 180,000 Daltons.
5. IgE:
IgE in normal serum is lowest amongst all other Igs. Its molecular weight is about 200,000 Daltons. It has also two L
chains and two H chains joined by one S-S bonds. Allergens stimulate the plasma cells for the synthesis of IgE
antibodies.
Igs are also further sub dived into subclasses. Such as IgG1, IgG2, IgG3 and IgG4 and each of them have a distinct
H chain. IgM has IgM1 and IgM2. IgA is also classified into IgA1 and IgA2.
Immunoglobulins are highly antigenic or immunogenic. Antibodies can be produced against different classes or
subclasses of Igs, after injecting them into another heterologus species. The different Igs differ in antigenic
determinants mainly on А-chains. The differences are mainly due to amino acid sequences.

18A.
ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and
quantifying substances such as peptides, proteins, antibodies and hormones.
1. Antibody coating. Specific capture antibody is immobilized on high protein-binding plates by overnight incubation. ...
2. Protein capture. Samples and standard dilutions are added to the wells and will be captured by the bound antibodies.
3. Detection antibody. ...
4. Streptavidin-enzyme conjugate. ...
5. Addition of substrate. ...
6. Analysis.

18B
Monoclonal antibodies (mAbs) are produced by introducing an antigen to a mouse and then fusing polyclonal B
cells from the mouse's spleen to myeloma cells. The resulting hybridoma cells are cultured and continue to produce
antibodies to the antigen.
 1. 1. Immunization: The very first step in
hybridoma technology is to immunize
an animal (usually a mouse), with
appropriate antigen. ...
2. 2. Cell Fusion: ...
3. 3. Selection of Hybridomas: ...
4. 4. Screening the Products: ...
5. 5. Cloning and Propagation: ...
6. 6. Characterization and Storage:

19A.

Pathways leading to cell death. Healthy cells respond to death-inducing stimuli by initiating a variety of molecular
pathways leading to cell death. Completion of the proper pathway is a critical cellular function to ensure that the
appropriate outcome is ultimately achieved in a multicellular organism. Failure to die in response to particular stimuli
can result in abortive embryogenesis and organ dysfunction and contributes to the initiation of cancer.
Proinflammatory death is vital in triggering appropriate immune responses or, in the extreme, may cause tissue
pathology and organ dysfunction. Therefore, pathway utilization can dramatically influence biological systems.
Apoptosis is a pathway leading to cell death that features the activation of initiator caspases that activate effector
caspases to cleave cellular substrates. Apoptotic cells demonstrate cytoplasmic and nuclear condensation, DNA
damage, formation of apoptotic bodies, maintenance of an intact plasma membrane, and exposure of surface
molecules targeting intact cell corpses for phagocytosis. In the absence of phagocytosis, apoptotic bodies may
proceed to lysis and secondary or apoptotic necrosis. Autophagy features degradation of cellular components within
the intact dying cell in autophagic vacuoles. The morphological characteristics of autophagy include vacuolization,
degradation of cytoplasmic contents, and slight chromatin condensation. Autophagic cells can also be taken up by
phagocytosis. Oncosis is the prelethal pathway leading to cell death accompanied by cellular and organelle swelling
and membrane breakdown, with the eventual release of inflammatory cellular contents. Pyroptosis is a pathway to
cell death mediated by the activation of caspase-1, a protease that also activates the inflammatory cytokines, IL-1β,
and IL-18. This pathway is therefore inherently proinflammatory. Pyroptosis also features cell lysis and release of
inflammatory cellular contents. Undoubtedly, other pathways exist that have not yet been described.
Necrosis is when cells die accidentally due to, say, trauma (ex. a poisonous spider bite), or lack of nutrients (ex. lack
of blood supply). Necrosis begins with cell swelling, the chromatin gets digested, the plasma and organelle
membranes are disrupted, the ER vacuolizes, the organelles break down completely and finally the cell lyses,
spewing its intracellular content and eliciting an immune response (inflammation).

19B.
Type I Hypersensitivity:
Type I hypersensitive reactions are the commonest type among all types which is mainly induced by certain type of
antigens i.e. allergens. Actually anaphylaxis means “opposite of protection” and is mediated by IgE antibodies
through interaction with an allergen.
(i) Mode of Action:
During the activity, this class of antibody (IgE) binds with high affinity to F C (Fragment crystalized) receptors on the
surface of constant domains of tissue mast cells and blood basophils. Such IgE-coated mast cells and basophils are
said to be sensitized. When the individual is exposed to the same allergen again, then it cross-links the membrane
bound IgE on sensitized mast cells and basophils and degranulation of those cells result (Fig. 11.3).

(ii) Biological effects:

1. Normally anaphylactic responses are of a mild type producing symptoms— like hay-fever, running nose, skin erup-
tions called as ‘nives’ or breathing difficulties.
2. The pharmacologically active mediators released from the granules exert biological effects on the surrounding
tissues.
3. In some cases, the responses may be severe, develop within a few minutes (2-30 mins) and may even cause
death before any medical help is called anaphylactic shock.
4. The principal effects of vasodilation and smooth muscle contraction may be either systematic or localized.
(iii) Components of type-I reactions:
There are different types of components which are required for type-1 reactions:
1. Different allergens
2. Reaginic antibody (IgE)
3. Mast cells and basophils
4. IgE—binding FC receptors.
5. High—affinity and low-affinity receptors.
(iv) Therapy for Type-I hypersensitivity:
1. The first step in controlling type I is to identify the offending allergen and avoid contact if possible.
2. Removal of house pets, dust-control measures.
3. Repeated injections of increasing doses of allergens called hypo sensitization.
4. Enhancement of phagocytosis by IgG antibody which is referred to a blocking antibody because it competes for the
allergens, binds and forms a complex that can be removed by phagocytosis.
5. Successful use of anti-histamine drugs result better with respect to type I hypersensitivity.

20. A

Transplantation involves the removal of damaged/injured tissues or organs from the body of a person and their
substitution by similar tissues/organs from a donor.
Organs which are transplanted:
Transplants of organs that are now feasible include bone marrow, lungs, heart, liver, cornea and also kidneys as
written above.
Types of organ transplantation:
Organ transplantation is of four types.
1. Auto-graft:
It is grafting of one’s own tissue to another part of the body, e.g., skin graft. It is most successful transplantation.
2. Iso-graft:
It is transplantation from a twin brother or sister i.e., donor and recipient are genetically identical.
3. Allograft:
It is the transplantation between individuals of same species, but with different genetic background.
4. Xeno-graft:
It is transplantation between animals of different species. Tissue matching, blood group matching are essential before
undertaking any graft/ transplant. Transplantation may result in the rejection of transplanted organs. The immune
system recognizes the protein in the transplanted tissue or organs as foreign and initiates cellular immunity. To
suppress the immune response during transplantation, histocompatibility antigen and immunosuppresants play an
important role.
(i) Histocompatibility is the property of having the same or mostly the same alleles of a set of genes called the major
histocompatibility complex. The major histocompatibility complex (MHC) is a set of molecules displayed on cell
surfaces that are responsible for lymphocyte recognition and antigen presentation. It is encoded by several genes
located on human chromosome 6. Major histocompatibility complex (MHC) is also referred as the HLA (or Human
Leucocyte Antigen) System in humans.
(ii) Immunosuppressive drugs or immunosuppressant’s are drugs that are used to prevent rejection. A kidney
transplant from an identical twin is always successful. If kidney is transplanted from other person except twin is also
successful with the use of an immunosuppressant. The drug, named cyclosporine is a good immunosuppressant.
Cyclosporine A is produced by Trichoderna polysporum. It destroys T-cell mediated immune responses, while spares
humoral antibody responses. This drug prevents rejection of kidney, heart and liver transplants.

20B.
The principle of immunisation or vaccination is based on the property of ‘memory’ of the immune systems. Vaccines
also generate memory-B and T cells that recognise the pathogen quickly. In snake bites the injection which is given
to the patients contains preformed antibodies against the snake venom. This type of immunisation is called passive
immunisation.
The process of introduction of vaccine into an individual to provide protection against a disease is called vaccination.
In vaccination, a preparation of antigenic proteins of pathogens or inactivated/weakened pathogens (vaccine), is
introduced into the body.
These antigens generate the primary immune response, and the memory В and T cells. When the vaccinated person
is attacked by the same pathogen, the existing memory T or В cells recognise the antigen quickly and attack the
invaders with a massive production of lymphocytes and antibodies.
Vaccination and immunisation are two different processes. Vaccination only refers to the administration of a vaccine
or toxoid, while immunisation is the process by which the body produces antibodies against the vaccine preventable
diseases through administration of specific vaccines. These are used to protect us and our domestic animals against
viral and bacterial diseases.
Attenuated whole-agent vaccines use living but attenuated (weakened) microbes. Examples of attenuated vaccines
are the Sabin polio vaccine and those used against measles, mumps and rubella (MMR). The widely used vaccine
against the tuberculosis bacillus and certain of the newly introduced, orally administered typhoid vaccines contain
attenuated bacteria.
2. Inactivated whole-agent vaccines use microbes that have been killed. Inactivated virus vaccines used in humans
include those against rabies, influenza and polio (the Salk polio vaccine). Inactivated bacterial vaccines include those
for pneumococcal pneumonia, cholera, pertussis (whooping cough) and typhoid.
3. Toxoids which are inactivated toxins, are vaccines directed at the toxins produced by a pathogen. Examples.
Vaccines against tetanus and diphtheria.
4. Subunit vaccines use only those antigenic fragments of a microorganism that best stimulate an immune response.
Subunit vaccines that are produced by genetic modification techniques, meaning that other microbes are
programmed to produce the desired antigenic fraction, are called recombinant vaccines. For example, the vaccine
against the hepatitis В virus consists of a portion of the viral protein coat that is produced by genetically modified
yeast.
5. Conjugated vaccines have been developed in recent years to deal with the poor immune response of children. The
polysaccharides are combined with porteins such as diphtheria toxoid. This approach has led to the very successful
vaccine for Haemophilic influenza type b, which gives significant protection.
6. Nucleic acid vaccines or DNA vaccines are among the newest and most promising vaccines, although they have
not yet resulted in any commercial vaccine for humans. Experiments with animals show that plasmids of “naked” DNA
injected into muscle results in the production of the protein encoded in the DNA. The “gene gun” method for injecting
nucleic acids into plant cells
These proteins stimulate an immune response. A problem with this type of vaccine is that the DNA remains effective
only until it is degraded. Indications are that RNA, which could replicate in the recipient, might be a more effective
agent.
The secondary immune response:
Memory cells may remain in the body for decades. Every new encounter with the same antigen results in a rapid
proliferation of memory cells. This is also called “booster response”. The antibody titer after subsequent encounters is
far greater than during a primary response and consists mainly of IgG antibodies. This accelerated, more intense
response is called the secondary immune response. Antibodies produced during a secondary response have an even
higher affinity for the antigen.
A person who had been suffering from diseases like measles, small pox or chicken pox becomes immune to
subsequent attacks of these diseases. It includes spleen, lymph nodes, tonsils, Peyer’s patches of small intestine and
appendix.
The increased power and duration of the secondary immune response explain why immunization (method of
providing immunity artificially, it is called vaccination) is usually accomplished by injecting antigen in multiple doses.