Antigen and antibody interaction is the basis of serological testing. There are several types of serological tests that detect this interaction, including precipitation, agglutination, hemagglutination, enzyme-linked immunosorbent assay (ELISA), Western blot, hemagglutination inhibition, and immunofluorescence. These tests exploit the formation of antigen-antibody complexes to diagnose diseases, identify pathogens, and detect proteins.

1. Antigen and Antibody Interaction
By
basis of serological testing
Dr. Humera Kausar
(PhD Molecular Biology)

2. Antigen Antibody Complex
Antigen and Antibody
Interaction is the basis
of serological testing

4.  Principle
Precipitation
Soluble antigen combine with its soluble
antibody and form a lattice that develops
into a visible precipitate.
 One of the easiest of serological tests
 Occur best when antigen and antibody are
present in optimal proportions
(Equivelance).

5. Precipitation
Examples;
• Ring precipitation test
• A solution of antigen is layered on the
surface of the antibody in a small tube
or capillary tube. A narrow ring of
precipitate occurs near the junction of
two fluids.
• This type of test is used for grouping
streptococci (according to C
polysaccharide) and for determining
unknown proteins in forensic
medicine

6. Gel - diffusion precipitation
• Antigen and antibody meet in an agar medium and a thin line
of precipitate is produced there (antigen - antibody complex).
• 1. Single diffusion
Antigen diffuses in the agar medium (antibody is homogenously
spread in the agar). It is carried out either in the tubes - single
gel - diffusion by Oudin or on the slide - single radial
immunodiffusion by Mancini.

7. Gel - diffusion precipitation
The principle of the reaction: The antigen is placed in a well cut
in an agar gel containing suitable diluted antibody. A ring of
precipitate forms where the reactants meet in optimal
proportions. The higher is the concentration of the examined
antigen, the greater is the diameter of the ring. According to the
diameter of the ring it is possible to count the concentration of
the examined antigen.

8. Gel - diffusion precipitation
This type of immunodiffusion is used for quantitative
determination of immunoglobulins (IgM, IgG, IgA and
IgD), complement components and other serum proteins.

9. Gel - diffusion precipitation
2. Double immunodiffusion
is used more often. Antigen and antibody are
allowed to diffuse towards each other in an agar
medium, e.g. from separate wells cut in an agar
plate or in a Petri dish. When antigen and antibody
meet in optimal proportions they produce a thin
line of precipitate. Position of the precipitate line
depends on concentrations of both antigen and
antibody and on their diffusion coefficient.
•

10. Gel - diffusion precipitation
• This reaction is used for diagnosing various bacterial,
viral, fungal and autoimmune diseases and for
recognizing toxin production by Corynebacterium
diphtheriae.

11. Agglutination

12. Agglutination
6

13. Some of the major differences between Agglutination
reaction and Precipitation reaction are:
 The size of the antigen is different in these reactions.
 Antigens are soluble molecules in precipitation reactions
while they are large and insoluble molecules in case of
agglutination.
 Agglutination is a more sensitive reaction in comparison to
precipitation.

15. Haemagglutination
15
RBC

16. Viral Hemagglutination
• the attachment of viral particles by their receptor sites
16
to more than 1 cell.
• As more and more cells become attached in this
manner agglutination becomes visible

18. Equivalence point:
(suitable proportion between the virus particles and
18
RBCs)

19. Hemagglutination test: method
Titer = 32 HA units/ml
1:8
1:2 1:2 1:2 1:2 1:2
8 16 32 64 128 256
virus
serial dilution
mix with red
blood cells
side view
top view
One HA unit :minimum amount of virus that causes
complete agglutination of RBCs

20. 20

22. ELISA
 The ELISA plate is coated with Antibody to
detect specific antigen

23. Enzyme labeled
Alkaline Phosphatase (AP)
or
Horseradish Peroxidase (HRP)
They are readily available
They are stable and remain usefull for long

24. Types of ELISA

25. Disease
Normal

27. Sandwich ELISA Procedure
 Prepare a surface to which a known quantity of
capture antibody is bound.
 Block any non specific binding sites on the surface
 Apply the antigen-containing sample to the plate.
 Wash the plate, so that unbound antigen is
removed.
 Apply enzyme linked primary antibodies as
detection antibodies which also bind specifically to
the antigen.
 Wash the plate, so that the unbound antibody-enzyme
conjugates are removed.

28. Sandwich ELISA Procedure
Apply a chemical which is converted by the
enzyme into a coloured product.
Measure the absorbency of the plate wells to
determine the presence and quantity of antigen

29. Indirect ELISA Procedure
• The protein antigen to be tested for is added
to each well of ELISA plate, where it is given
time to adhere to the plastic through charge
interactions
• A solution of non-reacting protein is added to
block any plastic surface in the well that
remains uncoated by the protein antigen

30. Indirect ELISA-Cont
 Then the serum is added, which contains a
mixture of the serum antibodies, of unknown
concentration, some of which may bind
specifically to the test antigen that is coating
the well.
 Afterwards, a secondary antibody is added,
which will bind to the antibody bound to the
test antigen in the well. This secondary
antibody often has an enzyme attached to it

31. Indirect ELISA-Cont
 A substrate for this enzyme is then added. Often,
this substrate changes colour upon reaction with
the enzyme. The colour change shows that
secondary antibody has bound to primary
antibody, which strongly implies that the donor
has had an immune reaction to the test antigen.
 The higher the concentration of the primary
antibody that was present in the serum, the
stronger the colour change. Often a spectrometer
is used to give quantitative values for colour
strength

32. Indirect ELISA

33. Western blot
The western blot (sometimes called the protein immunoblot) is a
widely accepted analytical technique in molecular biology used to
detect specific proteins in the given sample.
Used for the detection of HIV

34. Western blot

35. Western blot

36. Western blot

37. HEMAGGLUTINATION INHIBITION
37
TEST (HI)
VIRUSE SERUM

38. In the absence of anti-virus antibodies
38
Erythrocytes
Virus
Virus agglutination of
erythrocytes

39. In the presence of anti-virus antibodies
39
Erythrocytes
Virus Anti-virus
antibodies
Viruses unable to bind to
the erythrocytes

40. 40
Hemagglutination Inhibition

41. Immunoflouresence
This technique uses the specificity of antibodies to their
antigen to target fluorescent dyes to specific biomolecule
targets within a cell, and therefore allows visualization of the
distribution of the target molecule through the sample.

42. Immunoflouresence

44. THANKS