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Ag-Ab interactions are widely studied because of their importance in diagnosing infections mainly viral infections.
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ANTIGEN ANTIBODY INTERACTIONS
Presented By,
Iqra Altaf
Jinnah University For Women
IntroductionSimilar 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.
Affinity &
Avidity
Types
Compliment Fixation Test
ELISA
Immunofluorescence
Agglutination Test
COMPLEMENT FIXATION TEST
CFT ‒ Used to detect the presence of
either specific antibody or specific
antigen in a patient's serum.
Co
mp
lim
ent
Fix
ati
on
Tes
t
Is a biochemical technique used mainly in
immunology to detect the presence of an
antibody or an antigen in a sample.
ELISA
IMMUNOFLUORESCENCE
INTRODUCTION
Immunofluorescence is the labeling ofantibodies or antigens with fluorescent dyesImmunofluorescent labeled tissue sections
are studied using a fluorescence microscope.Coons et al (1942) showed that labelled
dyes can be conjugated to Ab’s and theselabelled antibodies can be used to detectAg’s.
Dyes Used Commonly :
FLUORESCEIN
An organic dye that is the most widely used labelfor immunofluorescence procedure absorbslight(490nm) and emits an intense yellow greenfluorescence(517nm).
PHYCOERYTHRINIs an efficient absorber of light(30 fold greater thanfluorescein) and a brilliant emitter of red fluorescence,stimulating as a wide used in immunofluoresence.
TYPES OF IMMUNOFLUORESCENCE:-
‰ Direct immunofluorescence ‰ Indirect immunofluorescence
Direct Immunofluorescence: Used to detect antigen in clinicalspecimens using specificfluorochrome labeled antibody.Ag is fixed on the slideFluorescein labeled Ab’s are layeredover itSlide is washed to removeunattached Ab’sExamined under UV light in anfluorescent microscopeThe site where the Ab attaches toits specific Ag will show apple greenfluorescence
Indirect Immunofluorescence Indirect test is a double-
layer technique The unlabelled antibody
is applied directly to thetissue substrate
Treated with afluorochrome-conjugatedanti-immunoglobulinserum
Advantage Over Direct IF:Because several fluorescent anti-immunoglobulins can bind to each antibodypresent in the first layer, the fluorescence isbrighter than the direct test.
More time-efficient since it is only one signallabelled reagent, the anti-immunoglobulin, isprepared during the lengthy conjugationprocess.
AGGLUTINATION TEST
Agglutination ReactionWhen 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.
Qualitative Agglutination TestAgglutination tests can be used in a qualitative mannerto assay for the presence of an antigen or an antibody. Theantibody is mixed with the particulate antigen and apositive test is indicated by the agglutination of theparticulate antigen.For example, a patient's red blood cells can be mixedwith antibody to a blood group antigen to determine aperson's blood type. In a second example, a patient'sserum is mixed with red blood cells of a known bloodtype to assay for the presence of antibodies to that bloodtype in the patient's serum.
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
Prozone EffectOccasionally, 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.
Passive HemagglutinationSimilar 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.
Hemagglutination Inhibition
Measures the ability of soluble antigen toinhibit the agglutination of antigen-coatedred blood cells by antibodies.
A fixed amount of antibodies to theantigen in question is mixed with a fixedamount of red blood cells coated with theantigen .
Also included in the mixture are differentamounts of the sample to be analyzed forthe presence of the antigen.
If the sample contains the antigen, thesoluble antigen will compete with theantigen coated on the red blood cells forbinding to the antibodies, therebyinhibiting the agglutination of the RBCs.