classes of immunoglobulins, types of antibodies and their production

1. ANTIBODY

2. Antibody
 A protien formed in
response to antigen.
 Belong to a group of
proteins called
immunoglobulins (Ig)
 The pivotal reagent
common to all
immunohistochemical
techniques.
 Soluble
immunoglobulin of

3. History
 Initially, Antibodies were identified only by
their electrical charge
 Since the 1890s, immunologists have known
that the molecules of humoral immunity are
present in serum.
 In 1939, Tiselius and Kabat performed
electrophoretic studies with rabbit antiserum
specific for ovalbumin.
 They found that electrophoresis of serum from
unimmunized rabbits resolved serum proteins
into four dominant families of differing mobility.

4.  The albumins were the fastest-migrating
(most negatively charged) fraction.
 They followed by the alpha- (a), beta- (b), and
gamma- (g) globulins.

5.  Then hyperimmunized rabbits with hen
ovalbumin to get a strong antibody
response.
 The electrophoresed the immune serum,
they observed a large increase in the
gamma-globulin fraction.
 Hence concluded that antibodies were
gamma-globulins.

6. The Structure
 Called as monomer
due to simple
structure.
 Consists of two
chains of
polypeptides, each
linked to each other
by disulphide bonds.

7.  Composed of two identical
heavy chains (H) and two
identical light chains (L).
 H chain with a molecular
weight of 50,000 daltons
and L chain of 25,000.
 Covalent interchain
disulfide bridges join L to H
and H to H chains to form a
Y-shaped molecules.
 The end portion of the Y’s
arm called variable(V)
region.
 The stem is called constant
(C) or Fc region.

8.  The H chains differ in antigenic and
structural properties, which determine the
class and subclass of the molecule.
 The two L chains are either of type kappa
(κ) or lambda (λ) and the distribution of this
differs in all Ig classes, subclasses, between
different species.
 By participating in the tertiary structure, they
confer greater stability and can assume T
shape.

10. The chemical structure of antibodies
explains three functions of antibodies:
 Binding versatility
 Binding specificity and
 Biological activity.

11. Immunoglobulins comprise five
major classes
 IgG
 IgA
 IgM
 IgD
 IgE
In decreasing quantity found in plasma or
serum

12. Monomer
IgD, IgE, IgG
Dimer
IgA
Pentamer
IgM

13. The most frequently utilized
antibodies in immunohistochemistry
 IgG
 IgM

14. IgG
 Most common in serum.
 Accounts to 80% of the total i.e,10-20mg in
1ml of serum.
 The heavy chains of IgG are denoted as
gamma (γ) chains.
 Molecular Weight = 150 kDa
 IgG has the general formula of γ2 κ2 or γ2 λ2.

15.  It is composed of two γ heavy chains, and
two light chains of either type κ or type λ.
 On the basis of antigenic differences, IgG
can be classsified as IgG1, IgG2, IgG3 and
IgG4.
 Within two weeks after injection, they
usually predominate.
 Have a mean survival of approximately
three weeks.

17. Role
 Readily crosses the walls of blood vessels
and enter tissue fluids.
 Eg: Maternal IgG can cross the plcenta and
confer passive immunity to the foetus
 Protects from circulating bacteria and virus.
 Neutralize bacterial toxins.
 Trigger the complement system.
 Enhances the effectiveness of phagocytic
cells.

18. IgM
 Makes up to 5% of antibody serum.
 General formula expressed as (μ2 κ 2)5 or (μ 2
λ 2)5.
 MW approximately 900 kDa, five subunits of
approximately 180 kDa each.
 Each subunit is linked by a sulfhydryl-rich
peptide, the J chain (15 kDa).
 The J-chains contribute to the integrity and
stability of the pentamer.

20.  IgM is the first humoral antibody detectable
on the surface of develoing ß-lymphocytes.
 Secreted to blood during primary antibody
responsse.
 Has a total of 10 antigen binding sites along
with J chain.
 Have a relatively short half-life of only four to
six days.

21.  Treatment of pentameric IgM with 0.1%
mercaptoethanol cleaves the disulfide
bridges.
 Subclasses
• IgM1
• IgM2

22. Role
 Agglutinates bacteria
 Activates complement
 Enhances ingestion of pathogens by
phagocytic cells.
 Special antibodies like red blood cells
agglutinins and heterophile antibodies.

23. IgA
 Constitutes about 10-12%.
 Molecular weight of 60,000 Daltons.
 Proteins of respiratory, gastrointestinal
mucous membrane and external body
secretion.
 Predominant Ig in secretions - milk, saliva,
tears, mucus.
 Exists as monomer, dimer and polymer.
 Prevent the attachment of pathogens

25. IgA transport across cell (Transcytosis)

26. IgD
 Make up to only 0.2%
 Molecular weight is 1,80,000 daltons.
 Acts as a primer receptors for specific
antigens on foetal lymphocytes.
Functions
 Regulates the synthesis of other Ig
 Foetal antigen receptors

27. IgE
 Makes up to only 0.002%.
 Binds tightly by the stem region to receptors.
 During reaction releases histamine and other
chemical mediation.
 Participate in immediate hypersensitivities
reations. Ex. Asthma, anaphylaxis, hives.
 Destruct the pathogens.

28. Cross-Linkage of Bound IgE Antibody with
Allergen Causes

29. Antibody production
 Produced by White blood cells i.e, B cells (B
lymphocytes) of stem cells in bone marrow.
 Activated in the presence of antigen and
produces plasma cells.
 Plasma cells create antibodies that are
specific to specific antigen.
 Once the infection is under control antibody
production decreases and circulates in lower
concentration.

30. Polyclonal Antibodies
 Heterogeneous mixture of antibodies directed
against various epitopes of the same antigen.
 Generated by different B-cell clones of the
animal.
 Rabbits are frequently the species of choice in
polyclonal antibody production

31. Polyclonal antibody production

32. Monoclonal antibodies
 Homogeneous population of immunoglobulin
directed against a single epitope.
 The antibodies are generated by a single B-
cell clone from one animal.
 Immunize Animal With Antigen
 Multiple Clones Are Generated.
 Good For In Vivo
 Most commonly produced in mice and rabbits.

33. Monoclonal antibodies production

34. Uses
 As antiviral antibodies. Eg: diagnosis of
influenza virus type for treating rabies.
 As a diagnostic reagent in clinical
biochemistry.
 For the purification of protein products.
 Therapeutically to overcome the unwanted
effects of immune system. Eg: Rejection in
kidney transplanting.