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Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) are direct binding assays for antibody or antigen and both work on the same principle, but the means of detecting specific binding is different. Radioimmunoassays are commonly used to measure the levels of hormones in blood and tissue fluids, while ELISA assays are frequently used in viral diagnostics. For both these methods one needs a pure preparation of a known antigen or antibody, or both, in order to standardize the assay. This assay will be described with a sample of pure antibody, but the principle is similar if pure antigen is used instead. In RIA for an antigen, pure antibody against that antigen is radioactively labeled, usually with 125I; for the ELISA, an enzyme is linked chemically to the antibody. The unlabeled component, which in this case would be antigen, is attached to a solid support, such as the wells of a plastic multiwell plate, which will absorb a certain amount of any protein.
The labeled antibody is allowed to bind to the unlabeled antigen, under conditions where nonspecific absorption is blocked, and any unbound antibody and other proteins are washed away.
Antibody binding to RIA is measured directly in terms of the amount of radioactivity retained by the coated wells, whereas in Elisa, binding is detected by a reaction that converts a colorless substrate into a colored reaction product. The color change can be read directly in the reaction tray, making data collection very easy, and ELISA also avoids the hazards of radioactivity. This makes ELISA the preferred method for more direct binding assays.
Labeled anti-immunoglobulin anti-bodies can also be used in RIA or ELISA to detect binding of unlabeled antibody to unlabeled antigen-coated plates. In this case, the labeled anti-immunoglobulin antibody is used in what is termed a ‘second layer’. The use of a second layer also amplifies the signal, as at least two molecules of the labeled anti-immunoglobin antibody are able to bind to each unlabeled antibody. RIA and ELISA can also be carried out with unlabelled antibody stuck on the plates and labeled antigen added.
The principle of the enzyme linked immunosorbent assay (ELISA)
To detect antigen A, purified antibody specific for antigen A is linked chemically to an enzyme. The samples to be tested are coated onto the surface of plastic wells to which they bind nonspecifically. Residual sticky sites on the plastic are blocked by adding irrelevant proteins (not shown). The labeled antibody is then added to the wells under conditions where non specific binding.