For Nursing students

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