The document outlines various antigen-antibody interactions and their significance in immunoassays. It discusses several techniques, including the complement fixation test, ELISA, immunofluorescence, and agglutination tests, detailing how they work and their applications. Different types of immunofluorescence methods and agglutination reactions, including qualitative and quantitative tests, along with concepts like the prozone effect and hemagglutination inhibition, are also explored.

1. ANTIGEN ANTIBODY
INTERACTIONS
Presented By,
Iqra Altaf
Jinnah University For Women

2. Introduction
Similar to an enzyme substrate interaction
Interactions between the antigenic
determinant, or epitope, of the antigen and
the variable-region of the antibody
molecule.
High specificity lead to development of
various immunoassays.

3. Affinity &
Avidity

4. Types
Compliment Fixation
Test
ELISA
Immunofluorescence
Agglutination Test

5. COMPLEMENT FIXATION TEST
CFT ‒ Used to detect the presence of
either specific antibody or specific
antigen in a patient's serum.

6. Compliment Fixation Test

7. ELISA
Is a biochemical technique used mainly in
immunology to detect the presence of an
antibody or an antigen in a sample.

13. IMMUNOFLUORESCENCE

14. INTRODUCTION
Immunofluorescence is the labeling of
antibodies or antigens with fluorescent dyes
Immunofluorescent labeled tissue sections
are studied using a fluorescence microscope.
Coons et al (1942) showed that labelled
dyes can be conjugated to Ab’s and these
labelled antibodies can be used to detect
Ag’s.

15. Dyes Used Commonly :
FLUORESCEIN
An organic dye that is the most widely used label
for immunofluorescence procedure absorbs
light(490nm) and emits an intense yellow green
fluorescence(517nm).
PHYCOERYTHRIN
Is an efficient absorber of light(30 fold greater than
fluorescein) and a brilliant emitter of red fluorescence,
stimulating as a wide used in immunofluoresence.

16. TYPES OF
IMMUNOFLUORESCENCE:-
 ‰ Direct immunofluorescence
 ‰ Indirect immunofluorescence

17. Direct Immunofluorescence:
Used to detect antigen in clinical
specimens using specific
fluorochrome labeled antibody.
Ag is fixed on the slide
Fluorescein labeled Ab’s are layered
over it
Slide is washed to remove
unattached Ab’s
Examined under UV light in an
fluorescent microscope
The site where the Ab attaches to
its specific Ag will show apple green
fluorescence

18. Indirect Immunofluorescence
 Indirect test is a double-layer
technique
 The unlabelled antibody
is applied directly to the
tissue substrate
 Treated with a
fluorochrome-conjugated
anti-immunoglobulin
serum

19. Advantage Over Direct IF:
Because several fluorescent anti-immunoglobulins
can bind to each antibody
present in the first layer, the fluorescence is
brighter than the direct test.
More time-efficient since it is only one signal
labelled reagent, the anti-immunoglobulin, is
prepared during the lengthy conjugation
process.

20. AGGLUTINATION TEST

21. Agglutination Reaction
When a particular Ag is mixed with its Ab’s in the
presence of electrolytes at a suitable temperature and
Ph, the particles are clumped and agglutinated.
The Ab of the serum causes the cellular Ag to form
clumps and these are called agglutinins.
The particulate Antigens that are aggregated are called
agglutinogens.
 When the antigen is an erythrocyte the term hem
agglutination is used.

22. Qualitative Agglutination Test
Agglutination tests can be used in a qualitative manner
to assay for the presence of an antigen or an antibody. The
antibody is mixed with the particulate antigen and a
positive test is indicated by the agglutination of the
particulate antigen.
For example, a patient's red blood cells can be mixed
with antibody to a blood group antigen to determine a
person's blood type. In a second example, a patient's
serum is mixed with red blood cells of a known blood
type to assay for the presence of antibodies to that blood
type in the patient's serum.

24. Quantitative Agglutination Test
 Serial dilutions are made of a sample to be tested
for antibody and then a fixed number of RBCs or
bacteria or other such particulate antigen is added.
 Maximum dilution that gives agglutination is
determined.
Maximum dilution that gives visible agglutination
is called the titer.
Results are reported as the reciprocal of the
maximal dilution that gives visible agglutination

25. Prozone Effect
Occasionally, it is observed that when the
concentration of antibody is high, there is no
agglutination and then, as the sample is diluted,
agglutination occurs.
The lack of agglutination at high concentrations of
antibodies is called the prozone effect.
 Lack of agglutination in the prozone is due to
antibody excess resulting in very small complexes
that do not clump to form visible agglutination.

27. Passive Hemagglutination
Similar to haemagglutination
test but the physical nature of
reaction is altered.
Ag is coated on the surface of
a carrier particle and thereby
making the reaction more
sensitive.
The carrier particles used can
be RBC’s, latex particles and
bentonite.
Used for the diagnosis of
Rheumatoid arthritis.

28. Hemagglutination Inhibition
 Measures the ability of soluble antigen to
inhibit the agglutination of antigen-coated
red blood cells by antibodies.
 A fixed amount of antibodies to the
antigen in question is mixed with a fixed
amount of red blood cells coated with the
antigen .
 Also included in the mixture are different
amounts of the sample to be analyzed for
the presence of the antigen.
 If the sample contains the antigen, the
soluble antigen will compete with the
antigen coated on the red blood cells for
binding to the antibodies, thereby
inhibiting the agglutination of the RBCs.