This presentation clearly describes what are immunoglobulins, their types, structure and how they get diversified into different isotopes to fight with foreign antigens.

1. IMMUNOGLOBULINS
AND ITS DIVERSITY
By :-VANSHIKA SRIVASTAVA

2. IMMUNOGLOBULINS
 A protein produced by plasma cells and lymphocytes and characteristic of these types of cells.
 Immunoglobulins play an essential role in the body's immune system. They attach to foreign substances,
such as bacteria, and assist in destroying them.
 Immunoglobulins occur in two main forms:
 SOLUBLE ANTIBODIES
 MEMBRANE-BOUND ANTIBODIES.

3. FORMATION OF ANTIBODIES

4.  These two terms, antibodies and immunoglobulins, are used interchangeably but are different in
minimal aspects.
 Antibodies are specific glycoprotein configurations produced by B-lymphocytes and plasma cells in
response to a specific antigen and capable of reacting with that antigen.
 Immunoglobulins structurally similar animal proteins that may or may not be endowed with antibody
activity.
 Thus all antibodies are immunoglobulins but all immunoglobulins are not antibodies.
 The function of an antibody is to bind its antigen as tightly as possible and then direct it towards other
components of the Immune System so that it can be destroyed.
 Immunoglobulins are named based on the class, or subclass of the heavy chain and type or subtype of
light chain.
ANTIBODY(Ab) vs. IMMUNOGLOBULINS (Igs)

5. Antibodies production is the sole function of the B cells.
Not toxic or destructive, bind the pathogen tightly and target
destructive components of the immune system.
Antibodies are useful in the defense against extracellular
pathogens.
Antibodies are secreted in the secondary lymphoid organs and in
bone marrow and find their way to the extracellular spaces.
During the course of an infection antibody effectiveness
improves steadily.

6. SERUM PROTEIN LEVELS
Antigen
entry

7. HUMAN IMMUNOGLOBULIN CLASSES
1. IgG-Gamma (γ) heavy chains
Structure: Monomer
Percentage serum antibodies: 80%
Location: Blood, lymph, intestine
Half-life in serum: 23 days
Complement Fixation: Yes
Placental Transfer: Yes
Known Functions: Enhances phagocytosis ,neutralizes toxins and viruses.
Protects fetus and newborn, Compliment activation

8. 2. IgA-Alpha (α) heavy chains
Structure: Monomer, Dimer and Secretory
Percentage serum antibodies: 10-15%
Location: Secretions (tears, saliva, intestine, milk), blood and lymph. 40 mg of secretory
IgA /kg body weight is secreted through intestine
Half-life in serum: 6 days
Complement Fixation: No
Placental Transfer: No
Known Functions: Localized protection of mucosal surfaces. Provides immunity to
infant digestive tract.

9. 3. IgM-Mu (μ) heavy chains
Structure: Pentamer and monomer
Percentage serum antibodies: 5-10%
Location: Blood, lymph, B-cell surface (monomer)
Half-life in serum: 5 days
Complement Fixation: Yes
Placental Transfer: No
Known Functions: First antibodies produced during an infection. Effective against
microbes and agglutinating antigens.

10. 4. IgD-Delta (δ) heavy chains
Structure: Monomer
Percentage serum antibodies: 0.2%
Location: B-cell surface, blood, and lymph
Half-life in serum: 3 days
Complement Fixation: No
Placental Transfer: No
Known Functions: In serum function is unknown. On B-cell surface, initiate immune
response.

11. 5. IgE- Epsilon (ε) heavy chains
Structure: Monomer
Percentage serum antibodies: 0.002%
Location: Bound to mast cells and basophils throughout body.
Half-life in serum: 2 days
Complement Fixation: No
Placental Transfer: No
Known Functions: Allergic reactions. Possibly lysis of worms.

12. HUMAN IMMUNOGLOBULIN SUBCLASSES
 IgG SUBCLASSES
Four subclass:-
 IgG1-Gamma 1 (γ1) heavy chains
 IgG2-Gamma 2 (γ2) heavy chains
 IgG3-Gamma 3 (γ3) heavy chains
 IgG4-Gamma 4 (γ4) heavy chains

13.  IgA SUBCLASSES
Two subclasses:-
 IgA1-Alpha 1 (α1) heavy chains
 IgA2-Alpha 2 (α2) heavy chains

14.  Immunoglobulins are classified on the basis of type of light chains.
 Kappa (κ)
 Lambda (λ)
--It has four subtypes:-
 Lambda 2 (λ2)
 Lambda 1 (λ1)
 Lambda 3 (λ3)
 Lambda 4 (λ4)
• Heterogeneity
Immunoglobulins considered as a population of molecules are normally very heterogeneous because they are
composed of different classes and subclasses each of which has different types and subtypes of light chains. In
addition, different immunoglobulin molecules can have different antigen binding properties because of different
VH and VL regions.
TYPES OF IMMUNOGLOBULINS

15.  Immunoglobulin G (IgG) is the major class of antibody molecule and one of the most abundant proteins in
the blood serum.
 It has:-
 2 Heavy chains
 2 Light chains
 Variable regions  antigen binding
 Constant regions
 These two chains are linked by non-covalent and disulfide bonds into a complex of Mr 150,000.
 The heavy chains of an IgG molecule interact at one end, then branch to interact separately with the light
chains, forming a Y-shaped molecule.
 Each chain is made up of identifiable domains; some are constant in sequence and structure from one IgG
to the next, others are variable.
STRUCTURE OF IgG

16.  The constant domains have a characteristic structure
known as the immunoglobulin fold, a well-conserved
structural motif in the all class of proteins.
 There are three of these constant domains in each
heavy chain and one in each light chain.
 The heavy and light chains also have one variable
domain each, in which most of the variability in amino
acid residue sequence is found.
 The variable domains associate to create the antigen-
binding site.

17. RIBBON STRUCTURE OF IgG

18. STRUCTURE OF IgA
 Serum IgA is a monomer but IgA found in secretions is a
dimer.
 When IgA exits as a dimer, a J chain is associated with it and
also has another protein associated with it called the secretory
piece or T piece.
 sIgA is sometimes referred to as 11S immunoglobulin.
 The secretory piece helps IgA to be transported across
mucosa and also protects it from degradation in the
secretions.

19. STRUCTURE OF IgM
 IgM normally exists as a pentamer (19S
immunoglobulin) but it can also exist as a monomer.
 In the pentameric form all heavy chains are identical
and all light chains are identical.
 Thus, the valence is theoretically 10. IgM has an extra
domain on the mu chain (CH4) and it has another
protein covalently bound via a S-S bond called the J
chain.
 This chain functions in polymerization of the molecule
into a pentamer.

20. STRUCTURE of IgD
 Secreted IgD is produced as a monomeric antibody with
two heavy chains of the delta (δ) class, and two Ig light
chains.

21. STRUCTURE of IgE
 Monomers of IgE consist of two heavy chains (ε chain) and two light chains, with the ε chain containing 4
Ig-like constant domains (Cε1-Cε4).
 IgE is typically the least abundant
isotype.
 IgE also has an essential role in type I
hypersensitivity.

22. IMMUNOGLOBULIN SUPERFAMILY(IgSF)
 Large and functionally diverse group of proteins that share a common structural feature, the Ig fold.
 Comprised of individual domains of about 100 amino acids in length with a pair of conserved cysteine
residues forming a disulphide bond spaced some 50–70 amino acids apart.
 Anti-parallel β-strands, stabilized by a single disulphide bond.
 2% of genes fall into the IgSF in accordance with human genome sequence analysis.
 IgSF molecules are broadly expressed by many cell types, and their ancient evolutionary origin testifies to
the remarkable stability and versatility of the Ig fold.

23. STRUCTURE OF THE IMMUNOGLOBULIN SUPERFAMILY DOMAINS
 Consist of two β-sheets with a
total of 7 strands roughly arranged
to form a ‘Greek Key’ motif.

24.  There are two variants of fundamental C Ig folds; C1 and C2 with difference in association of β –
strands.
 Each strand consists of 5–10 amino acids with the
side chains of hydrophobic amino acids facing the
interior of the sandwich, and those of hydrophilic
amino acids facing outwards.

25.  V-type Ig folds have two additional short β-
strands, than C-type Ig folds, designated C′ and
C′′ for a total of nine β-strands.
 In C-type Ig folds cysteines are 55-60 amino acids
apart while in V-type Ig folds they are 65-75
amino acids apart.

26. FUNCTIONS OF IgSF
 ANTIGEN RECOGNITION BY ANTIBODIES AND T-CELL
RECEPTORS.
 CELLADHESION.
 SIGNAL TRANSDUCTION.

27. ENGINEERING OF Ig DOMAINS
 Expression of Ig domains on the surface of bacteriophage or yeast for selection of high-affinity
antibody or TCR reagents.
 Engineering of catalytic enzymes supported on the Ig core (catalytic antibodies) and monoclonal
antibodies expressed in bacteria, yeast, insect cells, mammalian cells and even in plants such as
tobacco.

28. ANTIBODY AFFINITY
 Measures the strength of interaction between an epitope and an antibody’s antigen binding site.
 It is defined by the same basic thermodynamic principles that govern any reversible biomolecular
interaction.
 High-affinity antibodies will bind a greater amount of antigen in a shorter period of time than low-
affinity antibodies.
 Affinity of monoclonal antibodies can be measured accurately because they are homogeneous and
selective for a single epitope.

29.  Gives a measure of the overall strength of an antibody-antigen complex.
 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.
 Multivalent nature of antibodies and multimeric interactions between antibody and antigen accounts
for stabilization of antigen-antibody complex.
ANTIBODY AVIDITY

30. ISOTYPES
 Distinct forms of light or heavy chains which are present in all members of a species, encoded at
distinct genetic loci.
 Kappa and lambda are isotypes of light chains.
 Mu (μ), delta (δ), gamma-1 (δ1), etc. are isotypes of heavy chains. Encoded by the constant region
segments of the immunoglobulin gene which form the fc portion of an antibody.
 Isotype expression reflects the maturation stage of a B cell. Naive B cells express IgM and IgD isotypes
with unmutated variable genes.
 Other antibody isotypes (IgG1-4, IgA1-2, IgE) occurs via a process of class-switch recombination
(CSR) after antigen exposure.

31. ALTERNATE SPLICING IN B-CELLS
 The simultaneous synthesis of IgM and IgD by a single B-cell is the only phenomena where a normal
cell simultaneously produces two types of immunoglobulins.
 It also shows that the mu and delta chains are produced from the same chromosome, and not from the
two different allelic copies.

32.  Relocation of already rearranged V/D/J complex from its original position near the Cμ gene to a position
close to one of the other heavy-chain C-regions.
 Results in a new transcription unit and the synthesis of a heavy chain with same V-region but a new C-
region.
 Does not change the specificity of antibody.
 Class switching is triggered by cytokines; the isotype generated depends on which cytokines are present
in the B cell environment.
ISOTYPE SWITCHING

34. WHY KNOWING THE ISOTYPE MATTERS?
 Determination of immune response after immunization.
 Determination of immunoglobulins deficiency disorder.
 Assessment of optimum purification techniques.

35.  Genetic variants within the C-region sequences of particular isotypes that are inherited in an allelic manner
("allelic type").
 Different members of a species will therefore differ from one another with respect to which particular
alleles of a given isotype they received from their parents.
 Thus allotype refers to the idea that each immunoglobulin has unique sequences particular to the
individual's genome that manifest in its constant region (normally).
 Km1 and Km2 are allotypes of humans kappa chains; G1m(4) and G1m(17) are allotypes of human
gamma-1 chains.
 The presence of particular allotypes, like isotypes, can be readily detected in those normal sera in which
they are present.
ALLOTYPES

36. IDIOTYPES
 An antigenic specificity (epitope) which distinguishes a particular combination of VH and VL (the antigen
recognition site) from all others because of the shared characteristic between a group of immunoglobulin or T-
cell receptor (TCR) molecules based upon the antigen binding specificity and therefore structure of
their variable region.
 The variable region of antigen receptors of T-cells (TCRs) and B-cells (immunoglobulins)
contain complementarity determining regions (CDRs) with unique amino acid sequences.
 Unlike isotypes or allotypes, particular idiotypes can generally be detected (with very rare exceptions) only in
sera from myeloma patients.

37. Antibody idiotype is determined by:
 Gene rearrangement.
 Junctional diversity.
 P-nucleotides (palindromic nucleotides at sites
of single-strand breaks).
 N-nucleotides.
 Somatic hyper mutations.

38. GENES BEHIND IMMUNOGLOBULINS
 The immunoglobulin heavy locus on chromosome 14, containing 9 genes.
 The immunoglobulin kappa (κ) locus on chromosome 2, containing 1 gene.
 The immunoglobulin lambda (λ) locus on chromosome 22, containing 4 genes.

39. ANTIBODY DIVERSITY
 Germ-Line Theory
 Somatic Mutation Theory
 Dreyer and Bennett’s Two-Gene Model
 Multigene Organization of Immunoglobulin Genes

40. MULTIGENE ORGANIZATION OF IMMUNOGLOBULIN GENES
 κ and λ light chains and the heavy chains are encoded by separate multigene families situated on different
chromosomes.
 In germ-line DNA, each of these multigene families contains several coding sequences, called gene
segments, separated by noncoding regions.
 Gene segments are rearranged by splicing of noncoding segments and the coding gene segments brought
together to form functional immunoglobulin genes.
 kappa and lambda light-chain families contain L (leader), V (Variable), J (Junctional), and C (Constant)
gene segments.
 The heavy-chain family contains these same families plus D (Diversity) region.

41. Lambda Chain Multigene Family
 97 amino acids of the lambda chain variable region corresponded to the nucleotide sequence.
 13 C-terminal amino acids of the variable region were not matching with the corresponding nucleotide
sequence.
 39-base pair gene segment encoded the remaining 13 amino acid.
 There are 31 functional V gene segments, 4 J segments and 7 C segments.
Kappa Chain Multigene Family
 Forty V gene segments, five J gene segments and single C gene segment.
 Contains 5’ upstream leader sequence.

42. Heavy Chain Multigene Family
 Encoded by four gene segments.
 An additional gene segment, D segment.
 Encodes the amino acids with in the third complementarity determining region (CDR3).
 In humans 51 VH gene segments have been identified which are located upstream from a cluster of 27
functional DH gene segments.
 Six functional JH gene segment followed by a series of CH gene segment.
 CH gene segments are arranged in a specific sequence in the order Cμ, Cδ, Cγ, Cε, Cα.

43. GENE ARRANGEMENT

44. GENE REARRANGEMENT
Variable Region Gene Rearrangements
 Occurs in bone marrow.
 Undergoes in specific sequence.
 Heavy chain variable region gene rearrange first then light chain.
Light Chain Gene Rearrangement

45. Heavy Chain Gene Rearrangement

46. MECHANISM OF DNA REARRANGEMENTS
 DNA Rearrangement by Recombination Signal Sequence (RSS)
 Joining of Various Gene Segment by Recombinases
• Deletional Joining
• Inversional Joining

47.  Recombination signal sequences (RSSs) are present in the flanking region of each germ-line V, D, and
J gene segment. One RSS is located 3’ end to each V gene segment and 5’ end to each J gene segment.
 One RSS is present on the both sides of each D gene segment.
 Provides signals for the recombination process that rearranges the gene.
 Combination of seven base pair long palindromic sequences (heptamer) and a nine base pair long AT
rich conserved sequence (nanomer) with an intervening spacer DNA of either 12 base pairs or 23 base
pairs length.
 These recombination sequences are recognized by specific recombinases.
DNA Rearrangement by Recombination Signal Sequence (RSS)

49. Joining of Various Gene Segment by Recombinases
 Takes place at the junctions between RSS and coding sequences.
 Catalyzed by specific enzymes collectively called V(D)J recombinases.
 Encoded by two genes (recombination activating genes) designated as RAG-1 and RAG-2.
 Done by two methods:-
 Deletional Joining
 Inversional Joining

50.  Deletional Joining  Inversional Joining Deletional Joining

51. MECHANISM OF ANTIBODY DIVERSITY
Multiple germ-line gene segments
Combinatorial V(D)J joining
Junctional flexibility
P region and N region nucleotide addition
Somatic hypermutation
Combinatorial association of light and heavy chains

52. 1. Multiple germ-line gene segments
2. Combinatorial V(D)J joining

53. 3. Junctional flexibility
 Joining of recombination signal sequence is precise
while the joining of the coding sequence is imprecise.
 Produce various codons combinations leading to
change in amino acid sequence which is actually
coded by the original DNA.
 Comes under the third complementarity determining
region (CDR3) in immunoglobulin heavy chain and
light chain DNA.

54. 4. P region and N region nucleotide addition
 Production of short sequence nucleotides at
the end of the coding sequence during V-J
or V-DJ joining by the cleavage of the
single strand of DNA at the junction of a
variable region gene segment and signal
sequence.
 Addition of complementary bases to this
strand gives P-Nucleotide addition
 TdT addition which gives N-Nucleotide
addition.

55. 5. Somatic hypermutation
 The variable region genes undergo point mutations on antigenic stimulation that further increases the
antibody diversity
 Process by which additional antibody diversity generated in the rearranged variable region genes by
point mutations
 Rearranged variable region genes located within a DNA sequence containing about 1500 nucleotides.
 Frequency of somatic hypermutation (10-3 per base pair per generation) is around hundred thousand fold
higher than the spontaneous mutation rate (10-8 per base pair per generation) in other genes.
 Mutations are nucleotide substitutions.
 Occur throughout the VJ or VDJ segment and fall within the CDRs.
 Exact mechanism of somatic hypermutation is not known.

56. 6. Combinatorial Association of Heavy and Light Chains
 Possible combinations of heavy and light chains are therefore also contributed in the antibody diversity.
 Human genome has the potential to generate 45000 heavy chain genes and 1100 light chains genes as a
result of variable region gene rearrangements.
 If any of the heavy chain combines with any of the light chain, the potential combinations of heavy and
light chain variable genes will be 4.95 x 107.
 Combination process is not completely random, because not all the variable gene segments are used at
the same frequency.

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