This presentation discusses the concept of the generation of antibody diversity through immunoglobulin(Ig) gene rearrangements. The mechanism of V-J rearrangements (light chain) and V-D-J rearrangements (heavy chain) is elaborated in detail along with the Ig constant region rearrangements, called class-switching. The factors dictating antibody diversity and antigen-antibody interactions have also been discussed briefly.

1. PRESENTATION
BY
TATHAGAT SAH
BTech. Biotechnology
URN – 1901181

2. Ab Gene Rearrangements &
Ag-Ab Interactions
Table of Contents
• Two-Gene One-Polypeptide Model
• Tonegawa and Hozumi’s Experiment
• Heavy and Light Chain Multi-Genes
• Variable Region Rearrangements
o V-J Rearrangements (Light Chain)
o V-D-J Rearrangements (Heavy Chain)
• Mechanism of Rearrangements
• Constant Region Rearrangements
o Class-Switching
• Antibody Affinity and Avidity

3. Two-Gene One-Polypeptide Model
• Developed by Dreyer and Bennet in 1965
• Two separate genes code for the Heavy and Light chains,
One codes for the V region and the other for the C region
• These two genes come together during at the DNA level to
form a continuous message that can be transcribed and
translated into a single Ig heavy or light chain.
• There are thousands of V genes in germ-line but only one
gene for the C region
• This model was in conjunction with the “The Somatic
Variation Theory”

4. Tonegawa (1976): Immunoglobulin gene rearrangement

7. Heavy and Light Chain Multi-Genes
Multi-gene Families
• Light Chains: V, J and C gene segments
• Lambda: Humans (30V, 4J and 7C genes)
• Kappa: Humans (40V, 5J and 1C genes)
• Heavy Chains: V, D, J and C gene segments
• Heavy Chains: Humans (50V, 25D, 6J and 8 C genes)
 In heavy chains, the V, D and J segments encode the
variable domain while the C segment encodes the
constant domain.
 In light chains, the V and J segments encode the variable
domain while the C segment encodes the constant
domain.

8. The heavy chain locus has multiple V (variable) segments,
multiple D (diversity) segments, multiple J (joining) segments
and multiple C (constant) segments.
During maturation, one of each V, D and J segment is
randomly “chosen” and used to encode the final antibody
molecule.

9. Germ-line configuration of the heavy chain locus (mice)

10. Variable Region Rearrangements

11. Heavy Chain Rearrangements and
RNA Processing Events

12. Mechanism of Rearrangements
• In V(D)J recombination, the DNA encoding a complete
antibody V-region is assembled from V, D, and J (heavy
chain) or V and J (light chain) segments that are initially
separated by many kilo-bases of DNA.
• Each developing B cell generates a novel pair of variable
region genes by recombination at the level of genomic
DNA.
• Recombination is catalysed by a set of enzymes, many of
which are also involved in DNA repair functions , and is
directed to the appropriate sites on the Ig gene by
recognition of specific DNA sequence motifs called
Recombination Signal Sequences (RSSs).

13.  What mechanism ensures correct joining of gene
segments during rearrangement of the heavy and light
chain loci?
• Recombination signal sequences - conserved sequences in
regions just upstream or downstream of gene segments.
• Consist of a conserved heptamer and nonamer with a 12
or 23 bp spacer.
• The one-turn /two-turn rule - (12/23 rule) - recombination
occurs only between a segment with a 12 bp spacer and a
23 bp spacer.

14. RSS – At 3’ of V genes, 5’ of J genes and at both sides in D
genes
Rule: 12 (1 turn) or 23 (two turn) base pairs with conserved
flanking heptamer and nonamer
Recombinant Signal Sequences (RSS)

16. Rearrangement of gene segments is mediated by the
RAG1/RAG2 (Recombination Activating Genes) enzyme
complex (V(D)J recombinases).
The RAG1/RAG2 complex recognizes the heptamer/nonamer
sequences and cuts one strand of the DNA.

18. The hairpin is cut at a
random site
Signal Joint
Coding Joint

19. Terminal deoxynucleotidyl transferase (Tdt)
An enzyme that randomly adds in nucleotides during joining
of coding gene segments.

20. The join is repaired
Coding Joint
Note: This random rearrangement can lead to both
PRODUCTIVE and NON-PRODUCTIVE gene
rearrangements

21. The final “gene” encoding the antibody produced by a B cell
(and T cells) consists of a number of different segments.
This process of recombination of different gene segments and
addition of P and N nucleotides ensures that an enormous
number of different antigen specificities are possible.
Generation of antibody diversity
1. Multiple germline V, D and J gene segments
2. Combinatorial V-J and V-D-J joining
3. Somatic hypermutation
4. Junctional flexibility
5. P-nucleotide addition
6. N-nucleotide addition
7. Combinatorial association of heavy and light chains

22. Class Switching
• Antigen stimulation of a B cells Antibodies with same
variable Heavy (VDJ) with any CH gene segment
• Process dependent on Switch Regions
• Switch Regions (2-3 kb) are located upstream
from each CH segment, except IgD (Cd)
• Process driven by cytokines:
– IL-4 IgM to IgG1 or IgE
– IFN-g IgM to IgG2a
• Players in regulation: 1) switch regions, 2) switch
recombinases, 3) cytokine signals

23. Antibody Affinity and Avidity
Antibody Affinity measures the strength of interaction
between an epitope and an antibody’s antigen binding site. It
is defined by basic thermodynamic principles :
KA = affinity constant, KA describes how much Ab-Ag complex
exists at the point when equilibrium is reached
[Ab] = molar concentration of unoccupied binding sites on
the antibody
[Ag] = molar concentration of unoccupied binding sites on the
antigen
[Ab-Ag] = molar concentration of the Ab-Ag complex

24. Antibody Avidity gives a measure of the overall strength of an
antibody-antigen complex. It is dependent on three major
parameters:
• Affinity of the antibody for the epitope
• Valency of both the antibody and antigen
• Structural arrangement of the parts that interact
All antibodies are multivalent e.g. IgGs are bivalent and
and IgMs are decavalent. The greater an immunoglobulin’s
valency (number of antigen binding sites), the greater the
amount of antigen it can bind.
Similarly, antigens can demonstrate multivalency because
they can bind to more than one antibody. Multimeric
interactions between an antibody and an antigen help their
stabilization.

25. Ag-Ab
Interactions

26. Precipitin reactions
The interaction of antibody with antigen in
solution may cause formation of an
insoluble lattice that will precipitate out of
solution.
This precipitate will only form if:
- The antibody is bivalent or polyvalent
- The antibody or antibody mixture can
bind to at least two different sites on the
antigen (either two different epitopes or
two identical epitopes)
Monoclonal antibodies are likely to be less
efficient at Immuno-precipitation than
polyclonal antibodies.

27. END
OF
PRESENTATION!