A SEMINAR ON IMMUNO-DIAGNOSTIC TECHNIQUES

PRECIPITATION REACTIONS In Fluids

In Gels RADIAL IMMUNODIFFUSION DOUBLE IMMUNODIFFUSION

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

RADIOIMMUNOASSAY First developed by S. A. Berson and Rosalyn Yalow in 1960 to determine levels of insulin-anti-insulin complexes in diabetics

RIA has been widely used to screen for the presence of the hepatitis B virus. It can also be used to detect the levels of most of our hormones, digitoxin or digoxin in patients receiving these drugs. It can also be used to detect the concentration of certain abused drugs. RIA can also be used for the detection of specific secretory protein tuberculin derived from Mycobacterium tuberculosis.

ELISA ELISA are created by coating the antigen or antibody on a suitable plastic. To complete the reaction, an enzymatic detection method with a color-forming substrate is required. ELISA can be either competitive or noncompetitive. ELISA is a standard tool for quantifying the antibody or antigen in a serum. ELISA PLATE

INDIRECT 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 ELISA In this technique, the antibody is immobilized on a microtiter well. Sample containing antigen is added and allowed to react with the immobilized antibody. Second enzyme linked antibody specific for a different epitope on the antigen is added. Substrate added and colored reaction product is measured.

COMPETITIVE ELISA

The ELISA, or the enzyme immunoassay (EIA), was the first screening test widely used for HIV because of its high sensitivity. In an ELISA, a person's serum is diluted 400-fold and applied to a plate to which HIV antigens are attached. If antibodies to HIV are present in the serum, they may bind to these HIV antigens. The plate is then washed to remove all other components of the serum. Pregnancy tests also use a sandwitch ELISA method. An enzyme immunoassay (EIA) for detection of serum antibodies in patients with typhoid fever was developed using Salmonella typhi outer membrane protein (OMP) preparations as antigen.

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