A SEMINAR ONA SEMINAR ON

IMMUNO-DIAGNOSTICIMMUNO-DIAGNOSTICTECHNIQUESTECHNIQUES

PRECIPITATION REACTIONSPRECIPITATION REACTIONS

In FluidsIn Fluids

In GelsIn Gels

DOUBLE IMMUNODIFFUSION

RADIAL IMMUNODIFFUSION

AGGLUTINATIONAGGLUTINATION

The interaction between antibody The interaction between antibody and a particulate antigen results in and a particulate antigen results in visible clumping called visible clumping called agglutination. Ab that produce such agglutination. Ab that produce such reaction are called agglutinins.reaction are called agglutinins.

RADIOIMMUNOASSAYRADIOIMMUNOASSAY First developed by S. A. Berson and Rosalyn Yalow in 1960 to First developed by S. A. Berson and Rosalyn Yalow in 1960 to

determine levels of insulin-anti-insulin complexes in diabeticsdetermine levels of insulin-anti-insulin complexes in diabetics

RIA has been widely used to screen for RIA has been widely used to screen for the presence of the hepatitis B virus. the presence of the hepatitis B virus.

It can also be used to detect the levels of It can also be used to detect the levels of most of our hormones, digitoxin or digoxin most of our hormones, digitoxin or digoxin in patients receiving these drugs. in patients receiving these drugs.

It can also be used to detect the It can also be used to detect the concentration of certain abused drugs. concentration of certain abused drugs.

RIA can also be used for the detection of RIA can also be used for the detection of specific secretory protein tuberculin specific secretory protein tuberculin derived from derived from Mycobacterium tuberculosis.Mycobacterium tuberculosis.

ELISAELISA

ELISA are created by ELISA are created by coating the antigen or coating the antigen or antibody on a suitable antibody on a suitable plastic. To complete the plastic. To complete the reaction, an enzymatic reaction, an enzymatic detection method with a detection method with a color-forming substrate color-forming substrate is required.is required.

ELISA can be either ELISA can be either competitive or competitive or noncompetitive. noncompetitive.

ELISA is a standard tool ELISA is a standard tool for quantifying the for quantifying the antibody or antigen in a antibody or antigen in a serum.serum.

ELISA PLATE

INDIRECT ELISAINDIRECT ELISA

Primary antibody is added to an antigen-coated microtiter well. After any free Ab is washed away, the presence of antibody bound to the antigen is detected by adding an enzyme conjugated secondary antiisotype antibody (Ab2) which binds to the primary antibody. Any free antibody is then washed away and a substrate for the enzyme is added and the colored reaction product is measured by spectrophotometric plate reader, which can measure the absorbance of all the wells.

SANDWICH ELISASANDWICH ELISA In this technique, the In this technique, the

antibody is antibody is immobilized on a immobilized on a microtiter well. microtiter well.

Sample containing Sample containing antigen is added and antigen is added and allowed to react with allowed to react with the immobilized the immobilized antibody. antibody.

Second enzyme linked Second enzyme linked antibody specific for a antibody specific for a different epitope on different epitope on the antigen is added. the antigen is added.

Substrate added and Substrate added and colored reaction colored reaction product is measured.product is measured.

COMPETITIVE ELISACOMPETITIVE ELISA

The ELISA, or the enzyme immunoassay (EIA), The ELISA, or the enzyme immunoassay (EIA),

was the first screening test widely used for HIV was the first screening test widely used for HIV because of its high sensitivity. because of its high sensitivity.

In an ELISA, a person's serum is diluted 400-In an ELISA, a person's serum is diluted 400-fold and applied to a plate to which HIV fold and applied to a plate to which HIV antigens are attached. If antibodies to HIV are antigens are attached. If antibodies to HIV are present in the serum, they may bind to these present in the serum, they may bind to these HIV antigens. The plate is then washed to HIV antigens. The plate is then washed to remove all other components of the serum. remove all other components of the serum.

Pregnancy tests also use a sandwitch ELISA Pregnancy tests also use a sandwitch ELISA method.method.

An enzyme immunoassay (EIA) for detection of An enzyme immunoassay (EIA) for detection of serum antibodies in patients with typhoid fever serum antibodies in patients with typhoid fever was developed using was developed using Salmonella typhiSalmonella typhi outer outer membrane protein (OMP) preparations as membrane protein (OMP) preparations as antigen. antigen.

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