Enzyme-Linked Immunosorbant Assay (Elisa)
The enzyme-linked immunosorbant assay, which is commonly abbreviated to ELISA, is a technique that promotes the binding of the target antigen or antibody to a substrate, followed by the binding of an enzyme-linked molecule to the bound antigen or antibody. The presence of the antigen or antibody is revealed by color development in a reaction that is catalyzed by the enzyme which is bound to the antigen or antibody.
Typically, an ELISA is performed using a plastic plate which contains an 8 x 12 arrangement of 96 wells. Each well permits a sample to be tested against a whole battery of antigens.
There are several different variations on the ELISA theme. In the so-called direct ELISA, the antigen that is fixed to the surface of the test surface is the target for the binding of a complimentary antibody to which has been linked an enzyme such as horseradish peroxidase. When the substrate of the enzyme is added, the conversion of the substrate to a colored product produces a darkening in whatever well an antigen-antibody reaction occurred.
Another ELISA variation is known as the indirect technique. In this technique a specific antibody recognizes the antigen that is bound to the bottom of the wells on the plastic plate. Binding between the antigen and the antibody occurs. The bound antibody can then be recognized by a second antibody, to which is fixed the enzyme that produces the color change. For example, in this scheme the first, or primary, antibody could be a rabbit antibody to the particular antigen. The so-called secondary antibody could be a goat-antirabbit antibody. That is, the primary antibody has acted as an antigen to produce an antibody in a second animal. Once again, the darkening of a well indicates the formation of a complex between the antigen and the antibodies.
The third variation of the ELISA is known as the capture or sandwich ELISA. As the names imply, the antigen is sandwiched between the primary and secondary antibodies. In this technique, the primary antibody is bound to the bottom of the wells, rather than the antigen. Then, the antigen is added. Where the bound antibody recognizes the antigen, binding occurs. A so-called blocking solution is added, which occupies the vacant antibody sites. Then, an enzyme-labeled secondary antibody is added. The secondary antibody also recognizes the antigen, but the antigenic recognition site is different than that recognized by the primary antibody. The result is that the antigen is sandwiched in between two bound antibodies. Again, a color reaction reveals the complex.
The ELISA procedure has many applications. The procedure can provide qualitative ("yes or no") and quantitative ("how much") information on a myriad of prokaryotic and eukaryotic antibodies. Serum can be screened against a battery of antigens in order to rapidly assess the range of antibodies that might be present. For example, ELISA has proven very useful in the scrutiny of serum for the presence of antibodies to the Human immunodeficiency virus.
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