The document discusses antibody diversity and the theories that account for it. It describes three main theories: 1) The Germ Line Theory, which proposes that all antibodies are coded by germ line genes. 2) The Somatic Mutation Theory, which holds that diversity is generated by mutation of a relatively small number of antibody genes. 3) The Gene Rearrangement Theory, which states that diversity results from rearrangement of variable region gene segments during B cell differentiation. The document also outlines several means of antibody diversification, including multiple gene segments and junctional diversity.

2. CONCEPT OF ANTIBODY DIVERSITY
Antibody diversity is the phenomenon of immense variability characteristic
of antibodies, which enables the immune system to react specifically
against the essentially unlimited kinds of antigens it encounters. Antibody
diversity is accounted for by three main theories:
1. THE GERM LINE THEORY.
2. THE SOMATIC MUTATION THEORY.
3. THE GENE REARRANGEMENT THEORY.

3. ANTIBODY DIVERSITY
• Our immune system has potential to generate thousands or millions of
type specificity. This phenomena is called antibody diversity.
• The human heavy chain variable region is constructed from the joining of
three gene segments, V (variable), D (diversity), and J (joining), whereas
the light chain variable gene is constructed by the joining of two gene
segments, V and J. There are multiple V, D, and J segments at the heavy
chain and light chain loci.

4. 1. THE GERM LINE THEORY
• A Theory to account for the high degree of antibody variability found in
population. All antibodies are coded by genes of Germ line cells.
• Germ line proponents proposed that the genome contributed by the germ
cells contains a large types of Immunoglobulin genes. Each antibody was
encoded in a separate germ line gene. But they were unable to explain the
genetic mechanism to account for Antibody diversity.
• The Germ-Line Theory suggests that every B lymphocyte has all the genes
for every type of immunoglobulin but transcribes only one.

5. 1. THE GERM LINE THEORY
• It holds that each antibody producing cell has genes coding for all possible
antibody specificities, but expresses only the one stimulated by antigen.
• For every kappa-chain V-region there exists one unique germ-line gene. A
particular antibody-forming cell selects one of these and expresses it in
unmodified form.
• This does not occur in most
species including humans,
mice and other organisms but
may occur in Elasmobranchs.

6. 1. THE GERM LINE THEORY

7. 2. THE SOMATIC MUTATION THEORY
• The somatic mutation theory, which holds that antibody producing cells
contain only a few genes, which produce antibody diversity by mutation.
• Genome posses a relatively small number of antibody genes and diversity
is generated by mutation and recombination of these genes during
somatic development.

8. 3. THE GENE REARRANGEMENT
THEORY
•The gene rearrangement theory, which holds that antibody diversity is
generated by the rearrangement of variable region gene segments during
the differentiation of the antibody-producing cells.
•In heavy chains, the V (variable), D(diversity) and J(joining) segments
encode the variable domain while the C segment encodes the constant
domain.
• In light chains, the V (variable)and J (joining) segments encode the
variable domain while the C segment encodes the constant domain.
•During maturation, one of each V, D and J segment is randomly “chosen”
and used to encode the final antibody molecule.

9. ANTIBODY DIVERSITY
• There are 7 means of Antibody diversification:
1. Multiple germ line segments: Multiple germ
line V, D, and J gene segments contribute to
the diversity of the antigen binding sites in
antibodies.
2. Combinatorial V(D)J joining: The contribution
of multiple germ-line gene segments to anti-
body diversity is magnified by the random
rearrangement of these segments in somatic
cells.

10. ANTIBODY DIVERSITY
• Junctional diversity is the diversification of variable region exons due to
this imprecise joining of the coding ends. First, a small number of nucleo-
tides are often deleted from the coding end by an unknown exonuclease.
Junctional diversity involves the potential addition of two types of nucleo-
tides, P‐nucleotides and N‐nucleotides.
3. P- nucleotides addition: The palindromic sequences that result from the
asymmetric cleavage and template‐mediated fill‐in of the coding joints
are referred to as P‐nucleotides.

11. 4. N nucleotide addition: N‐nucleotides are
generated by the nontemplated addition
of nucleotides to the coding ends, which
is mediated by the enzyme terminal
deoxynucleotidyl transferase (TdT).
• The addition of these nucleotides may,
as for other events in antibody gene
assembly, result in the generation of
receptor genes that are out of frame.
ANTIBODY DIVERSITY

12. 5. Junctional flexibility: It describes the DNA
sequence variations introduced by the
improper joining of gene segments during
the process of V(D)J recombination.
6. Somatic hypermutation: All the antibody
diversity described so far stems from
mechanisms that operate during formation
of specific variable regions by gene
rearrangement. Additional antibody diversity is generated in rearranged
variable region gene units by a process called somatic hypermutation.
ANTIBODY DIVERSITY

13. ANTIBODY DIVERSITY
7. Combinatorial H & L chain
association: The creation of
diversity in the immunoglobulin
repertoire through this joining of
various gene segments is known
as combinatorial diversity.
Additional diversity is created by
the pairing of different heavy
chains with different lambda or
kappa light chains.

14. ISOTYPES
Each antibody has only one type of (γ, or α, or μ, or ε, or δ) heavy chain and
one type of (k or λ) light chain. The structural differences in the constant
region of a heavy chain or light chain determine immunoglobulin (Ig) class
and sub-class, types and subtypes within a species. These constant region
determinants are called isotypic determinants or isotypes.

15. ALLOTYPES
•Although all members of a species inherit the same set of isotype genes,
multiple alleles exist for some of the genes.
•These alleles encode subtle amino acid differences. Products of allelic
forms of the same gene will have slightly different amino acid sequence in
the constant regions, which are known as allotypic determinants.
•The sum of the individual allotypic determinants displayed by an antibody
determines its allotype.
•It represent the genetically determined differences in antibodies between
people.

16. ALLOTYPES

17. IDIOTYPES
•VH and VL domains of an antibody constitute an antigen-binding site. This
variable region has different structural conformation owing to the presence
of different amino acids.
•These unique amino acid sequences present in the VH and VL domains of a
given antibody also serves as a set of antigenic determinants. Each
individual antigenic determinant of the variable region is referred to as an
idiotope.
•Each antibody will present multiple idiotopes; the sum of the individual
idiotopes is called the idiotype of the antibody.

18. IDIOTYPES
•Idiotypes are antibodies that recognize different specific epitopes. The
thing that determines the idiotype is way at the end of the variable region;
it’s composed of a bunch of different idiotopes (or combining sites).

19. INTRODUCTION OF HYBRIDOMA
TECHNOLOGY
• Hybridoma technology is a method for producing large numbers of
identical antibodies (also called monoclonal antibodies).
• This process starts by injecting a mouse with an antigen that provokes an
immune response. A type of white blood cell, the B cell, produces antibody
that bind to the injected antigen.
• These antibody producing B-cells are then harvested from the mouse and,
in turn, fused with immortal B cell cancer cells, a myeloma to produce a
hybrid cell line called a hybridoma, which has both the antibody-producing
ability of the B-cell and the longevity and reproductivity of the myeloma.

20. •The hybridomas can be grown in culture, each culture starting with one
viable hybridoma cell, producing cultures each of which consists of
genetically identical hybridomas which produce one antibody per culture
(monoclonal) rather than mixtures of different antibodies (polyclonal).
• The myeloma cell line that is used in this process is selected for its
ability to grow in tissue culture and for an absence of antibody synthesis.
• In contrast to polyclonal antibodies, which are mixtures of many
different antibody molecules, the monoclonal antibodies produced by
each hybridoma line are all chemically identical
INTRODUCTION OF HYBRIDOMA
TECHNOLOGY

21. INTRODUCTION OF HYBRIDOMA
TECHNOLOGY

22. INTRODUCTION OF HYBRIDOMA
TECHNOLOGY
1. Immunization of a mouse
2. Isolation of B cells from the
spleen
3. Cultivation of myeloma cells
4. Fusion of myeloma and B cells
5. Separation of cell lines
6. Screening of suitable cell lines
7. Invitro or in vivo multiplication
8. Harvesting

23. MONOCLONAL ANTIBODIES
• Monoclonal antibodies are antibodies
that are made by identical immune cells,
clones belonging to a single parent cell.
• Monoclonal antibodies have monova-
lent affinity and bind to one particular
epitope of an antigen.
• Antibodies can be produced in special-
ized cells through a method now
referred to as hybridoma technology.

24. METHOD
1. Immunization of a mouse:
• The antigen against which the antibody are produce inject in selected animal.
• B cell convert in plasma cell.
• Plasma cells are produce monoclonal antibody.
2. Isolation of B cell:
• Spleen is collected and plasma cell are isolate .
3. Myeloma cell are cultivated in bone marrow:
• They should not produce antibodies.
4. Fusion of myeloma cell and B cell:
• It can be done using electro fusion or polyethylene glycol.
• Five different type of cell are produced.

25. METHOD

26. METHOD
5. Selection cell lines:
• It is carried out in HAT (Hypoxanthine Aminopterin Thymidine) media.
• Nucleotides can be synthesized by two way
a) Salvage pathway
b) De novo pathway for cell division.
• As Aminopterin in the media ,it block the key enzyme dihydrofolate
reductase so de novo synthesis can not follow.
• To operate salvage pathway cell must have HGPRT(hypoxanthine-
guanine phosphoribosyl - transferase ) enzyme and also have
hypoxanthine and thymidine as precursor.

27. POLYCLONAL ANTIBODIES
•Polyclonal antibodies are antibodies that are secreted by different B cell
lineages within the body.
•They are a collection of antibodies that react against a particular antigen,
each binding to different epitopes.
•Polyclonal antibodies are produced in the appropriate donor animals.
•Usually, antigens are conjugated with an adjuvant before immunizing the
animals.
• Adjuvant are substances that increase the immunogenicity of the antigen,
reducing the amount of antigen required as well as stimulating specific
immunity to it.

28. POLYCLONAL ANTIBODIES

29. METHOD
•Preimmunize blood samples are collected to produce baseline IgG levels.
•The first two immunizations are done within 14 days.
•Later immunizations are spaced at intervals of 4-6 weeks to maximize the
antibody production.
•Blood samples are collected 10 days after the completion of immunization
program.
•The serum screened for presence of antibodies with specific activity to
antigen. Method such enzyme-linked immunosorbent assay (ELISA) can be
used for the activity testing.

30. POLYCLONAL ANTIBOD

31. POLYCLONAL ANTIBODIES

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