The enzyme-linked immunosorbent assay (ELISA) is widely used to detect and estimate the concentration of a protein in a sample. The protein to be detected is regarded as the antigen. Therefore, the first step is to make an antibody specific for the target protein. A detection system is then attached to the rear of the antibody. Usually this consists of an enzyme that generates a colored product from a colorless substrate. Alkaline phosphatase, which converts X-Phos to a blue dye, is a common choice. The samples to be assayed are immobilized on the surface of a membrane or in the wells of a microtiter dish (Fig. 6.15). The antibody plus detection system is added and allowed to bind. The membrane or microtiter dish is then rinsed to remove any unbound antibody. The substrate is added, and the intensity of color produced indicates the level of target protein.
A variety of modifications of the ELISA exist. Often binding and detection are done in two stages, using two different antibodies. The first antibody is specific for the target protein. The second antibody recognizes the first antibody and carries the detection system. For example, antibodies could be raised in rabbits to a series of target proteins. The second antibody, which recognizes rabbit antibodies, could be produced in sheep. These are called secondary antibodies and are often described as, for example, sheep anti-rabbit. The secondary antibody has the detection system, and because it will recognize any antibody made in a rabbit, it does not have to be reengineered for each different target protein. This allows the use of the same final antibody detection system in each assay even if different primary antibodies are used to identify different proteins.