Antibodies

Chalee R. Sienes-Reyes, RMT, MSMT

Professor, Med. Lab. Sci. Dept.,
San Pedro College, Davao City
 Outline
• Antibody Production
• Basic Structure of Immunoglobulin and
Function
• Polyclonal and Monoclonal Antibody
• Response Events in the Germline Center
• Effector Mechanisms of Humoral Immune
Response
 Affinity
Strength of the reaction between a single antigenic
determinant and a single Ab combining site

High Affinity Low Affinity

Ab Ab

Ag Ag

Affinity =  attractive and repulsive forces

 Avidity
• The overall strength of binding between an Ag
with many determinants and multivalent Abs

Keq = 104 106 1010

Affinity Avidity Avidity
 Immunologic Memory
Virgin lymphocyte pool

PRIMARY RESPONSE

effector cells memory cell pool

SECONDARY
RESPONSE
effector cells memory cell pool
Immunologically Naive
No previous
experience
No memory
Must be educated
 The Primary Immune Response

antibod lag log phase plateau decline

y titer

antigen time
challenge
 Class Switching

antibdy
titer

IgM IgG

time
Four phases of the primary response

• a lag phase where no antibody is detected

• a log phase in which the antibody titer rises
logarithmically
• a plateau phase during which the antibody titer
stabilizes
• a phase (decline) during which the antibody is
cleared or catabilized
Dynamics of Antibody Production
 Comparison of Primary and
Secondary Responses

• Time course
• Antibody titer
• Antibody class
• Antibody affinity
 • Opsonization
• Neutralization
Terms to • Complement activation
remember • Primary response
• Secondary/ anamnestic/
booster response
• Lag phase
• Log phase
• Plateau period
• Memory cells
• Class switching
• Affinity maturation/ change
in affinity with time
 Cellular Events
• Antigen is “processed” by T lymphocytes and
macrophages.
• Possess special receptors on surface.
• Termed “antigen presenter cell” APC.
• Antigen presented to B cell
 Basic Antibody Structure
• Two identical heavy chains
– Gamma
– Delta
– Alpha
– Mu
– Epsilon

• Two identical light chains

– Kappa
– Lambda
Basic Antibody Structure
Basic Structure of Immunoglobulins
 Papain Cleavage
• Breaks disulfide bonds at hinge region
• Results in 2 “fragment antigen binding” (Fab)
fragments.
• Contains variable region of antibody molecule
• Variable region is part of antibody molecule
which binds to antigen.
Papain Cleavage
 Pepsin
• Breaks antibody above disulfide bond.
• Two F(ab’)2 molecules
• The rest fragments
• Has the ability to bind with antigen and cause
agglutination or precipitation
Papain and Pepsin Cleavage
 IgG
• Most abundant
• Single structural unit
• Gamma heavy chains
• Found intravascularly AND extravascularly
• Coats organisms to enhance phagocytosis
(opsonization)
• Crosses placenta – provides baby with immunity for
first few weeks of infant’s life.
• Capable of binding complement which will result in
cell lysis
• FOUR subclasses – IgG1, IgG2, IgG3 and IgG4
 IgA
• Alpha heavy chains
• Found in secretions
• Produced by lymphoid tissue
• Important role in respiratory, urinary and
bowel infections.
• 15-10% of Ig pool
• Does NOT cross the placenta.
• Does NOT bind complement.
• Present in LARGE quantities in breast milk
which transfers across gut of infant.
 Secretory IgA
• Exists as TWO basic structural units, a DIMER
• Produced by cells lining the mucous membranes.
 IgM
• Mu heavy chains
• Largest of all Ig – PENTAMER
• 10% of Ig pool
• Due to large size restricted to intravascular space.
• FIXES COMPLEMENT.
• Does NOT cross placenta.
• Of greatest importance in primary immune response.
 IgE
• Epsilon heavy chains
• Trace plasma protein
• Single structural unit
• Fc region binds strongly to mast cells.
• Mediates release of histamines and heparin>allergic
reactions
• Increased in allergies and parasitic infections.
• Does NOT fix complement
• Does NOT cross the placenta
 IgD
• Delta heavy chains.
• Single structural unit.
• Accounts for less than 1% of Ig pool.
• Primarily a cell bound Ig found on the surface of B
lymphocytes.
• Despite studies extending for more than 4 decades, a
specific role for serum IgD has not been defined
while for IgD bound to the membrane of many B
lymphocytes, several functions have been proposed.
• Does NOT cross the placenta.
• Does NOT fix complement.
• Polyclonal antibody
– Antigens possess multiple epitopes
– Serum antibodies are heterogeneous,
• To increase immune protection in vivo
• To reduces the efficacy of antiserum for various in vitro
uses
– To response facilitates the localization, phagocytosis,
and complement-mediated lysis of antigen
– To have clear advantages for the organism in vivo
• Monoclonal antibody
– Derived from a single clone, specific for a
single epitope
– For most research, diagnostic, and therapeutic
purposes
1975, by Georges Köhler and Cesar Milstein
- Be awarded a Nobel Prize in1984
 Formation and Selection of Hybrid Cells

• Hybridoma: the B cell X myeloma cell

– To be produce by using polyethylene glycol (PEG) to fuse
cells
– The myeloma cells: immortal growth properties
– The B cells: to contribute the genetic information for
synthesis of specific antibody
– Selected by using HAT medium (hypoxanthine,
aminoprotein, and thymidine)
• Myeloma cells are unable to grow
• B cells are able to survive, but can not live for extended
periods
Two different pathways to synthesis nucleotide in mammalian cells

(Folic acid analog)

Myeloma cells used in hybridoma

technology are double mutants,
they lack the HGPRTase and lose
the ability to produce Ig
 (Most common screening
techniques are ELISA and RIA)

Low concentration
(1-20 ug/ml)
High concentration
(1-10 mg/ml)
 Human Monoclonal Antibodies

• Production of human monoclonal antibody

– There are numbers of technical difficulties
• The lack of human myeloma cells to exhibit immortal
growth, be susceptible to HAT selection, to not secrete
antibody, and support antibody production in the
hybridoma made with them
• Human B cell sometimes have immortality
• That is the difficulty of readily obtaining antigen-
activated B cells
 Human Monoclonal Antibodies

• To culture human B cells in vitro to produce

human monoclonal antibody

– Transplant human cells with immune response

into SCID mice (lack a functional immune system)
Clinical Uses for Monoclonal Antibodies
• Very useful as diagnostic, imaging, and therapeutic
reagents in clinical medicine
– Monoclonal antibodies were used primarily as in vitro
diagnostic reagents
– Radiolabeled monoclonal antibodies can also be used in
vivo detecting or locating
• Immunotoxins
– To compose of tumor-specific monoclonal antibodies
coupled to lethal toxin
– Valuable therapeutic reagent
 Affinity Maturation and the
Generation of Memory B Cells
 Germline Organization
Of
Human Immunoglobulin gene
 Germline Organization Of
Human Immunoglobulin gene
Genomic organisation of Ig genes
(No.s include pseudogenes etc.)
LH1-123
VH 1-123
DH1-27 JH 1-9 Cm

Lk1-132
Vk1-132
Jk 1-5 Ck

Ll1-105
Vl1-105 Cl1 Jl1 Cl2 Jl2 Cl3 Jl3 Cl4 Jl4
 Ig light chain gene rearrangement by somatic
recombination

Vk Jk Ck
Germline

Rearranged
1° transcript

Spliced mRNA
 Ig light chain rearrangement: Rescue pathway

There is only a 1:3 chance of the join between the V and J region being in
frame

Vk Jk Ck

Non-productive rearrangement

Light chain has a second chance to make

a productive join using new V and J elements

Spliced mRNA transcript

 Ig heavy chain gene rearrangement

VH 1-123 DH1-27 JH 1-9 Cm

Somatic recombination occurs at the level of DNA which can now be

transcribed
 The constant region has additional, optional exons

Cm
Primary transcript RNA AAAAA

Each H chain domain (& the

hinge) encoded by separate Secretion
Polyadenylation Polyadenylation
exons coding site (secreted) site (membrane)
sequence pAs pAm

Cm1 h Cm2 Cm3 Cm4

Membrane
coding
sequence
 Membrane IgM constant region

DNA Cm1 h Cm2 Cm3 Cm4

Transcription
pAm
1° transcript Cm1 h Cm2 Cm3 Cm4 AAAAA
Cleavage & polyadenylation
at pAm and RNA splicing
Cm1 h Cm2 Cm3 Cm4 AAAAA
mRNA

Protein Membrane coding sequence

encodes transmembrane
region
that retains IgM in the cell
membrane

Fc
 Secreted IgM constant region

DNA Cm1 h Cm2 Cm3 Cm4

Transcription
pAs
1° transcript Cm1 h Cm2 Cm3 Cm4 AAAAA
Cleavage polyadenylation at
pAs and RNA splicing
mRNA Cm1 h Cm2 Cm3 Cm4 AAAAA

Protein Secretion coding sequence

encodes the C terminus of
soluble, secreted IgM

Fc
 The constant region has additional, optional exons

Cm
Primary transcript RNA AAAAA

Each H chain domain (& the

hinge) encoded by separate Secretion
Polyadenylation Polyadenylation
exons coding site (secreted) site (membrane)
sequence pAs pAm

Cm1 h Cm2 Cm3 Cm4

Membrane
coding
sequence
 RNA processing
Primary transcript RNA
pAs
V D J8 J9 Cm1 h Cm2 Cm3 Cm4 AAAAA

mRNA V D J8 Cm1 h Cm2 Cm3 Cm4 AAAAA

The Heavy chain mRNA is completed by splicing the VDJ region to the C
region

The H and L chain mRNA are now ready for translation

VL JL CL AAAAA

VH DH JH h CH AAAAA
Somatic hypermutation
Nature Reviews Immunology 2, 688-698 (2002)
 Class Switching

• DNA
rearrangement
– Antigen
dependent
– Switch site
– Same VDJ
– TH cytokines

During B cell development, many B cells switch

from making one class of antibody to making
another, this process is called class switching.
Antibody Class Switching
Class switching in immunoglobulin heavy chain genes
 Class Switching Recombination

Class-switch recombination (CSR) of immunoglobulin

heavy chains is the genetic process by which a B cell
switches from the production of IgM to the
production of IgG, IgE or IgA.
Cellular interactions important for
IgE class-switch recombination
 Effector Mechanisms of
Humoral Immune Response
 Neutralization
Toxin receptors

Host cell
Bacterial toxins
Neutralization by antibody

Phagocytosis of
antibody-antigen
complex by
macrophage
Forming phagosome Fc receptor
 Opsonization

Extracellular
Opsonization
bacteria

Macrophage

Ingestion by macrophage

Digestion in lysosome
 Complement Activation

Bacteria in plasma

Lysis and
ingestion

C1
C1 C2 C4

C2 Complement
C4
activation
Digestion in lysosome
IgE