1. Immunoglobulins
Structure

2. Definitions
Immunoglobulin is a generic term that refers to a diverse group of molecules found in the blood and tissue fluids
They are soluble globulin molecules and they generally migrate in an electrophoretic field at or near the gamma globulin fraction
An antibody is an immunoglobulin molecule capable of binding specifically with a known substance (antigen)
Immunoglobulins are synthesized by B lymphocytes and terminally differentiated B lymphocytes or plasma cells

4. Introduction
The first to show that antibodies reside in the gamma globulin fraction were Kabat and Tiselius in 1939 when they performed serum electrophoresis and they called them immunoglobulins
From the broad electrophoretic peak, it is clear that a heterogeneous collection of immunoglobulin molecules with slightly different charges is present
This heterogeneity was one of the early obstacles in attempts to determine the structure of antibodies, since analytical chemistry requires homogeneous crystalizable compounds as starting proteins

5. Certain diseases (multiple myeloma and light chain disease) provided the solution
Recently the hybridoma technology has made available antibodies in pure form
Porter in 1959 treated antibody with papain and produced three fragments of equal size
Two of the fragments were found to retain the antibody’s ability to bind antigen specifically but could no longer precipitate the antigen from solution
These two fragments were referred to as Fab
The third fragment could be crystallized out of solution and called FC

6. Fragment “antigen-binding”
ENZYME DIGESTION OF IMMUNOGLOBULINS
Fragment “antigen-binding”
Papain
Fragment crystallisable
Pepsin

7. At about the same time, Edelman discovered that when the immunoglobulin molecule was extensively reduced of by mercaptoethanol, it fell apart into four chains, two identical heavy chains (53000 Daltons) and two identical light chains (22000 Daltons)
Noble prize was awarded for Porter and Edelman for revealing antibody structure

8. Antibody structure
LIGHT
HEAVY
H+L
Antibody +
dimer

9. Structure
An immunoglobulin is composed of four polypeptide (glycoprotein) chains; two light and two heavy chains linked by disulfide bonds
Five different types of heavy chains; α, , µ, δ, and ε.
Two types of light chains; kappa (k) and lambda(λ) with a ratio of 3:2
An individual B cell or immunoglobulin monomer expresses either k or λ but not both
Within an immunoglobulin molecule, the two heavy chains and light chains are identical

10. Schematic diagram of structure of Ig

11. Some immunoglobulin molecules are basic monomers and others are composed of multiple copies (dimers or pentamers) of identical immunoglobulin monomers
Both heavy and light chains can be divided into regions or domains, homologous portions of an immunoglobulin chain, each composed of approximately 110 amino acids and contains an intradomain disulfide bridge

12. Heavy and light chains are
folded into domains, each containing
about 110 a.a. & an intrachain disulfide bond
that forms a loop of 60 amino acids.
(b) The μ and Є heavy chains
contain an additional domain
that replaces the hinge region

13. Light chains contain two regions, a variable (VL) and a constant ( CL) domain
Heavy chains contain a single variable (VH) and multiple constant domains(CH1, CH2, CH3, CH4)
Variable regions in both heavy and light chains are so named because of the extensive variation in the amino acid sequence found in immunoglobulin molecules made by different cells
The amino acid sequence determines the conformational structure of VH and VL
The combination of a light variable and a heavy variable region forms a pocket that constitute the antigen-binding region of the immunoglobulin molecule

14. Hinge Region Made up predominantly of cysteine and proline residues
It permits flexibility between the two Fab arms of the Y shaped molecule
It allows Fab to open and close to accommodate binding to two epitopes separated by a fixed distance

15. Variable Domains
Three areas of hypervariability occur between less variable stretches called Framework regions
Because they bind specifically to epitopes, the hypervariable regions are termed complementarity -determining regions (CDRs) of the L and H chains; CDR1, CDR2, and CDR3

16. V D J
CDR1
CDR2
CDR3

17. Immunoglobulin Fragments
Fab: produced by papain cleavage (2 Fab)
FC: produced by papain cleavage
Fd: it is the heavy chain portion of an Fab fragment cleaved by papain
F(ab)2: it is the dimeric molecule produced by pepsin cleavage which fragments the FC
Fd─ : it is the heavy chain portion of an Fab fragment cleaved by pepsin

18. Immunoglobulin Classes
Five different classes or isotypes depending on heavy chain antigenicity
Monomers, dimers or pentamers
Monomers are divalent having two identical antigen- binding sites

19. General structures of the five major classes
of antibody

20. IgM
First to be produced in an immune response. M for macroglobulin, M. Wt. about 106 Daltons, sedimentation coefficient 19S, Has an extra CH domain
Cell surface bound monomer or secreted pentamer. Five basic units (pentamer) joined by a J chain
( Daltons) synthesized by B cells or plasma cells
Five antigen-binding sites instead of the expected valence of 10 due to conformational constraints imposed by polymerization
6-8% of serum immunoglobulins

22. IgG
Monomer, M. Wt , sedimentation coefficient 7S. Least anodic of all serum proteins
Four subclasses; IgG1, IgG2, IgG3, and IgG4
Predominant immunoglobulin in blood, lymph, CSF, and peritoneal fluid
72-80% of serum immunoglobulins

24. IgA
The major immunoglobulin in external secretions such as saliva, mucous, sweat, gastric fluid, and tears
Monomer (serum) or dimer (secretions), M.Wt , sedimentation coefficient 7S
Two subclasses; IgA1 and IgA2
13-19% of serum immunoglobulins
Serum IgA is predominantly monomeric and monomeric IgA1 accounts for about 90% of serum IgA

25. Secretory Ig A
Secretory Ig A has a J chain and a secretory piece ( Daltons) which is synthesized by epithelial cells to facilitate passage of secretory IgA into mucous secretions and to protect it from cleavage (secretory IgA is more resistant than serum IgA)
Secretory IgA: - IgA1 90% in secretions above
the diaphragm
- IgA2 10% in lower GI

27. Structure and formation of secretory lgA

28. IgD Monomer, M. Wt. 180.000, sedimentation coefficient 7S
A major surface component of many B cells but present in very low concentrations (< 1% of serum immunoglobulins) in serum where it has no function.
It is not secreted by plasma cells and it is uniquely susceptible to proteolytic cleavage

29. IgE Monomer, M. Wt 200.000, sedimentation coefficient 8S
It has an extra CH domain
Less than 0.001% of serum immunoglobulins
Binds with high affinity to mast cells and basophils (Homocytophilic)

30. Antigenic determinants of immunoglobulins

31. Allotypes
Allelic forms of the same protein as a result of the presence of different forms of the same gene at a given locus
Allotypic differences at known loci usually involve changes in only one or two amino acids in the constant region of a chain
Important genetic markers inherited as dominant traits; Gm on  chain, Km (previously Inv) on kappa chains, and Am on α chain
The genes encoding the markers are expressed codominantly, so that an individual may be homozygous or heterozygous for a given marker

32. Ideotypes The antigenicity of the variable region of Fab
May or may not block binding of the antibody to the antigen depending whether produced against CDRs or Framework sequence. An ideotype represents an “internal immage” of the epitope
Public or cross reacting epitopes are ideotypes on different antibodies produced against the same epitope. Private epitopes react with only a particular antibody molecule
Regulation of an Immune response (Jerne’s Theory)