Procedure For The Enzyme-Linked Immunosorbent Assay

An enzyme-linked immunosorbent assay (ELISA) is used to detect the presence of an antigen in a sample. The antigen is immobilized to the well of a plate by adsorption or captured with a bound, antigen-specific antibody.

Antibodies to detect the antigen complex are then added if any. The detecting antibody can be covalently bound to an enzyme or it can be detected alone by an enzymatically bound secondary antibody.

An enzyme substrate is then added to the well which produces a visible signal related to the amount of antigen as measured by a spectrophotometer. You can also browse to know more about ELISA.

ELISA was carried out on polystyrene plates consisting of 96 wells or 384 wells. The reagents in the ELISA assay are immobilized, which facilitates the execution of the procedure. This assay has a monoclonal antibody layer on the microtiter plate.

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The preferred antibody is IgG, which is purified and used in conjugates to avoid interference by other proteins in binding enzymes. When a blood sample is added, a specific antibody (primary antibody) attaches to the protein of interest (eg a cytokine).

Secondary monoclonal antibodies bind to different epitopes on the protein. The assay is labeled with biotin, which allows subsequent protein binding, e.g. Streptavidin conjugated enzymes.

The enzymes often used in this procedure are horseradish peroxidase (HRP) and alkaline phosphatase (AP). All unbound reagents/serum components were removed by washing the plaque thoroughly. PBS-T (phosphate-buffered saline with Tween) was used as a diluent to remove unbound molecules.

A chromogenic substrate such as tetramethylbenzidine (TMB) was used for staining. It is added to the assay that obtains color from the enzymatic reaction (directly proportional to the amount of antigen bound). The choice of substrate depends on the type of equipment used (spectrophotometer, fluorometer, and luminometer).