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

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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.