This document discusses immunoglobulins and antibodies. It describes how immunoglobulins are Y-shaped proteins composed of light and heavy chains that bind to antigens. The five major classes of immunoglobulins are IgG, IgA, IgM, IgE, and IgD. IgG is the most abundant and mediates secondary responses, IgA is found in secretions, IgM acts as the primary antibody, IgE mediates allergic reactions, and the function of IgD is uncertain. ELISA techniques can be used to detect antigens or antibodies in samples using enzyme conjugates and colorimetric detection.

2. ANTIGEN
• Any substance which evokes an immunological
response is called antigen (Ag) or immunogen
• Epitope: specific site on antigen to which
antibody binds

3. • Proteins which specifically combine with
antigen
ANTIBODY

4. IMMUNOGLOBULINS (Ig)
• Glycoproteins (4-18% carbohydrate)
• (γ globulins)
• Constitute 20% of plasma proteins
• Produced by plasma cells
• Synthesis stimulated by particular antigen

5. NORMAL ELECTROPHORETIC PATTERN
γ β α1α2 Alb
Point of application
_ +

6. IMMUNOGLOBULINS
• Immunoglobulins constitute a heterogeneous
family of serum proteins, which either function
as antibodies or are chemically related to
antibodies

7. • All antibodies are immunoglobulins but all
immunoglobulins are not antibodies
IMMUNOGLOBULINS

8. STRUCTURE OF Ig
• Y shaped molecules
• Consists 2 heavy chains & 2
light chains
• Linked by disulphide bridges

9. IMMUNOGLOBULIN
STRUCTURE
• Light chain mol
wt 25K
• Heavy chain
mol wt 50K -70K
• Linked by
disulphide
bridges

10. IMMUNOGLOBULIN
STRUCTURE
LIGHT CHAINS
• Either kappa (κ)
or lambda (λ)
but not both
• One constant &
one variable
domain in each
chain

11. IMMUNOGLOBULIN
STRUCTURE
HEAVY CHAINS
• Either
α/µ/γ/δ/ε
• One variable &
3 constant
domains
• Hinge region
allows better fit
with Ag surface

12. VARIABLE REGION
• Variable region of
both light chain &
heavy chain
constitute Ag binding
site
• No two variable
regions are identical
IMMUNOGLOBULIN
STRUCTURE

13. CONSTANT REGION OF HEAVY CHAIN
• Complement binding & activation
• Binding to cell surface receptors
• Based on differences in their amino acid
composition heavy chains are classified as
α/µ/γ/δ/ε types
IMMUNOGLOBULIN STRUCTURE

14. CONSTANT REGION OF LIGHT CHAIN
• No known biological function
• Based on their differences of amino acid
composition kappa & lamda types
IMMUNOGLOBULIN STRUCTURE

15. CARBOHYDRATE
is
• Co-valently
bound to Fc
fragment
• Probably
protects Ig from
metabolic
degradation

16. Papain
Fc
Fab
IMMUNOGLOBULIN STRUCTURE
Cleaved by
papain
• at hinge
region
NH3
+
NH3
+
COO-

17. Pepsin
Fc Peptides
F(ab’)2
IMMUNOGLOBULIN STRUCTURE
NH3
+
NH3
+
COO-
Cleaved by
pepsin
• at hinge
region

18. IMMUNOGLOBULIN G (Ig G)
• Most abundant: 75-80%
• Half life: 20 days
• Also called 7S immunoglobulin
• Monomeric
• Can be 22 or 22

19. • Antibody in secondary response
• Opsonise bacteria - for phagocytosis
• Fixes complement - for bacterial killing
• Neutralize bacterial toxins and virus
• Cross placenta- Leads to Rh isoimmunisation
• Major protective antibody in new born
IMMUNOGLOBULIN G (Ig G)

20. • Second most abundant class (20%)
• Half life: 6-8 days
• 11S Ig
• Two forms
– Monomeric
– Dimeric
IMMUNOGLOBULIN A (Ig A)

21. • Secretory IgA formed by joining two
monomeric units at their carboxyl terminal by
J chains
• Additional secretory piece produced in liver
stabilize the dimer from proteolytic enzymes
IMMUNOGLOBULIN A (Ig A)
J Chain
Secretory Piece

22. • Secretory IgA present in secretions of GIT,
nose, pharynx, saliva, sweat etc
• Prevents attachment of bacteria and virus to
mucous membrane
• Monomeric IgA found in internal secretions
(synovial, amniotic, pleural, CSF)
IMMUNOGLOBULIN A (Ig A)

23. • 10% of immunoglobulins
• Half life 10 days
• Macroglobulin – (19S Ig)
• Pentamer: 5 units joined together by J chain
• Can bind with 5 antigens simultaneously
IMMUNOGLOBULIN M (Ig M)

24. • Primary antibody response against Bacteria
• Can fix complement
• Doesn’t cross placenta
• Called natural antibody as it is produced
without any known antigenic stimulus
–Eg:- Blood group antibodies
IMMUNOGLOBULIN M (Ig M)

25. • Trace amounts (0.004%)
• Half life: 2 days
• Sedimentation coefficient: 8s
• Monomeric
• Mostly extra vascular
IMMUNOGLOBULIN E (Ig E)

26. • Chiefly produced in the lining of respiratory
epithelium.
• Greatly raised in allergic reactions
IMMUNOGLOBULIN E (Ig E)

27. • Responsible for anaphylactic reactions
• Mediates immediate hypersensitivity
• Release histamine and slow reacting
substance from mast cell & basophil
IMMUNOGLOBULIN E (Ig E)

28. • Defends against worm infections
– Release enzymes from eosinophils
• Main host of defence against helminths
• Doesn’t fix complement
IMMUNOGLOBULIN E (Ig E)

29. • Very low concentration
• Half life: 3 days
• 7 S Ig
• Monomeric
IMMUNOGLOBULIN D (Ig D)

30. • Found on surface of many B cells and in serum
• May act as antigen receptor
• Function is uncertain
IMMUNOGLOBULIN D (Ig D)

31. IMMUNOGLOBULIN DISORDERS
• Hypergammaglobulinemia: increased Ig
• Hypogammaglobulinemia: decreased Ig

32. IMMUNOGLOBULINS
• Most predominant Ig
• Ig synthesized earliest in fetus
• Ig mediating anaphylactic reactions
• Ig in primary immune response
• Ig in secondary immune response
Ig G
Ig M
Ig E
Ig M
Ig G

33. • Ig in secretions
• Ig which crosses placenta
• Natural antibody
• Anti-Rh antibodies are
Ig A
Ig G
Ig M
Ig G
IMMUNOGLOBULINS

34. ELISA
• ELISA : enzyme-linked immunosorbent assay
• It is a commonly used laboratory test to detect
antigens or antibodies in the blood.
• The technique uses the formation of antigen
antibody complexes
• The antibodies are tagged with an enzyme
• When the substrate is added to it a colored
product is formed
• The color of the product is measured using
spectrophotometer

35. Indirect ELISA
• Antibody can be detected or quantitatively
determined by indirect ELISA.
• In this technique, antigen is coated on the
microtiter well.
• Serum sample containing the antibody is added
to the microtiter well and allowed to react with
the coated antigen.

36. • The antibody that is bound to antigen is
detected by adding an enzyme conjugated
secondary antibody that binds to the primary
antibody.
• A specific substrate for the enzyme is added.
• Enzyme hydrolyzes the substrate to form
colored products.

37. SANDWICH ELISA
• Antigens can be detected by sandwich ELISA.
• In this technique, antibody is coated on the
microtiter well.
• A sample containing antigen is added to the
well and allowed to react with the antibody
attached to the well, forming antigen-antibody
complex.

38. • A second enzyme-linked antibody is added
and allowed to react with the bound antigen.
• Finally substrate is added to the plate which
is hydrolyzed by enzyme to form colored
products

40. USES of ELISA
• ELISA is a technique used to estimate or detect
antigens or antibodies like:
• P 24 antibody testing for screening of HIV
• Beta HCG testing for pregnancy card test
• Estimation of hormones like insulin, Thyroid
hormones