Both antibody and antigen are usually multivalent.
Antigens carry multiple epitopes and stimulate production of antibodies which
bind to many of them, with different affinities.
How well antibody binds antigen is measured by two quantities
Affinity: the equilibrium constant of the reaction of Fab fragments of a
monoclonal antibody with an antigen containing one copy of epitope to
which the antibody binds. Such simple situations are never encountered in
the body and almost never in the lab.
Avidity: is a “pseudo-equilibrium constant” for a real, complex, antigen
carrying many different epitopes, in multiple copies, that binds to real,
multivalent,antibody molecules.
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
Affinity
Avidity
Avidity
Cross Reactivity
• The ability of an individual Ab combining site to
react with more than one antigenic determinant.
• The ability of a population of Ab molecules to
react with more than one Ag
Cross reactions
Anti-A
Ab
Anti-A
Ab
Anti-A
Ab
Ag A
Ag B
Ag C
Shared epitope
Similar epitope
Factors Affecting Measurement of
Ag/Ab Reactions
• Affinity
• Avidity
Ab excess
Ag excess
• Ag:Ab ratio
Equivalence – Lattice formation
A schematic drawing of a typical antibody molecule.
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It is composed of four polypeptide chains two identical heavy chains and
two identical light chains. The two antigen-binding sites are identical,
each formed by the N-terminal region of a light chain and the N-terminal
region of a heavy chain. Both the tail (Fc) and hinge region are formed by
the two heavy chains.
Immunoglobulin domains.
The light and heavy chains in an antibody
molecule are each folded into repeating
domains that are similar to one another.
The variable domains (shaded in blue) of
the light and heavy chains (VL and VH)
make up the antigen-binding sites.
The constant domains of the heavy chains
(mainly CH2 and CH3) determine the
other biological properties of the
molecule.
Antibody hypervariable regions.
Highly schematized drawing of how the three hypervariable regions in each light
and heavy chain together form the antigen-binding site of an antibody molecule
The folded structure of an IgG antibody molecule, based on x-ray
crystallography studies
The structure of the whole protein is shown in the middle, while the structure of a
constant domain is shown on the left and of a variable domain on the right. Both
domains consist of two b sheets, which are joined by a disulfide bond (not shown).
Note that all the hypervariable regions (red) form loops at the far end of the variable
domain, where they come together to form part of the antigen-binding site
Both light and heavy chains are made up of repeating segments each about 110
amino acids long and each containing one intrachain disulfide bond.
These repeating segments fold independently to form compact functional units
called immunoglobulin (Ig) domains.
A light chain consists of one variable (VL) and one constant (CL) domain
These domains pair with the variable (VH) and first constant (CH1) domain of
the heavy chain to form the antigen-binding region.
The remaining constant domains of the heavy chains form the Fc region, which
determines the other biological properties of the antibody.
The similarity in their domains suggests that antibody chains arose during
evolution by a series of gene duplications, beginning with a primordial gene
coding for a single 110 amino acid domain of unknown function.
A look into the secondary structure
Tabular Overview
Chains
15C8:H
15C8:L
Residues
217
213
Mol. Weight [D]
23064
23224
Chain Type
Protein
Protein
The assignments are: H=helix; B=residue in isolated beta bridge; E=extended beta strand;
G=310 helix; I=pi helix; T=hydrogen bonded turn; S=bend.
Chain 15C8:H
Compound
Igg 5C8
Type
Protein
Molecular Weight
23064
Number of Residues 217
Number of Alpha
0
Content of Alpha 0.00
Number of Beta
20
Content of Beta 47.47
Sequence and secondary structure
1 EVQLQQSGAE LVKPGASVKL SCTASGFNIK DTYMHWVKQK PEQGLEWIAQ
EEEE E EE TT EEE EEEEESS GG GSEEEEEEE TTS EEE EE
51 IDPANGNTKY DPKFQGKATI TADTSSNTAY LHLSSLTSED SAVYYCAADP
EETTTTEEEE TTTBTTEEE EEETTTTEEE EEE S GGG EEEEEEEE
101 PYYGHGDYWG QGTTLTVSSA KTTPPSVYPL APGSAAQTNS MVTLGCLVKG
SSTTS B
EEEEE
B EEEEE
S SSSS EEEEEEEES
151 YFPEPVTVTW NSGSLSSGVH TFPAVLQSDL YTLSSSVTVP SSTWPSETVT
EESS EEEE GGGTB SSEE E EESSSS EEEEEEEEE TTTTTTS E
201 CNVAHPASST KVDKKIV
EEEEEGGGTE EEEEE
The Generation of Antibody Diversity
Human antibody preimmune repertoire is made of more than 1012 different antibody molecules
• preimmune repertoire

low affinity*
• repeated stimulation by antigen

much higher affinity**
How can an animal make more antibodies than there are genes in its genome?
(human genome < 50,000 genes)
joining separate gene segments together before they are transcribed

mammalian immune system almost unlimited number of different light and heavy chains
(light and heavy chains of antibodies usually form the antigen-binding site)
*the antigen-binding sites of many antibodies can cross-react
with a variety of related but different antigenic determinants
** antigen stimulation greatly increases the antibody arsenal
The organization of the DNA sequences that encode the
constant region of an antibody heavy chain.
The coding sequences (exons) for each domain and for the hinge region are
separated by noncoding sequences (introns). The intron sequences are removed by
splicing the primary RNA transcripts to form mRNA. The presence of introns in
the DNA is thought to have facilitated accidental duplications of DNA segments
that gave rise to the antibody genes during evolution.
Antibodies are produced from three pools of gene segments
1. encodes heavy chains, 2. encodes k light chains, 3. encodes l light chains
IgM
IgD
IgG
IgA
IgE
Catene pesanti
m
d
g
a
e
Catene leggere
kol
kol
kol
kol
kol
% dell’Ig nel
sangue
5
<1
80
15
<1
In each pool gene segments that code for different parts of the variable region of
the light or heavy chains are brought together by site-specific recombination during
B cell development
Example: the V-J joining process involved in making a human k light chain
During the development
of a B cell, the randomly
chosen V gene segment
(V3 in this case)
is moved to lie precisely
next to one of the J gene
segments (J3 in this
Not yet expressed, and
therefore not rearranged,
antibody genes  a cluster
of five J gene segments
separated from the C-region
exon by a short intron and
from the 40 V gene segments
by thousands of bps.
These mRNAs are then translated
case).
The
"extra" J gene
segments (J4 and J5) and
the intron sequence are
transcribed (along with the
joined V3 and J3 gene
segments and the C-region
exon) and then removed by
RNA splicing to generate
mRNA molecules in which
the V3, J3, and C sequences
are contiguous.
The human heavy-chain gene-segment pool
There are 51 V segments, 27 D segments, 6 J segments, and an ordered cluster of
C-region exons, each cluster encoding a different class of heavy chain.
The D segment (and part of the J segment) encodes amino acids in the most variable part
of the V region.
The total length of the heavy chain locus is over 2 megabases.
Each C region is encoded by multiple exons
The genetic mechanisms involved in producing a heavy chain are the same as those shown
for light chains except that two DNA rearrangement steps are required instead of one.
• First a D segment joins to a J segment
• then a V segment joins to the rearranged DJ segment
Markers of Non-Self
Foreign molecules, too, carry distinctive
markers, characteristic shapes called epitopes
that protrude from their surfaces.
One of the remarkable things about the
immune system is its ability to recognize many
millions of distinctive non-self molecules, and
to respond by producing molecules such as
these antibodies—and also cells—that can
match and counteract each one of the non-self
molecules.
Any substance capable of triggering an
immune response is known as an antigen. An
antigen can be a bacterium or a virus, or even
a portion or product of one of these organisms.
Tissues or cells from another individual also
act as antigens; that's why transplanted tissues
are rejected as foreign.
Markers of Self
At the heart of the immune response is the
ability to distinguish between self and
nonself.
Every body cell carries distinctive
molecules that distinguish it as "self."
Normally the body's defenses do not
attack tissues that carry a self marker;
rather, immune cells coexist peaceably
with other body cells in a state known as
self-tolerance.