The enzyme-linked immunosorbent assay (ELISA), also known as an enzyme immunoassay (EIA), is a common serological test for the presence of proteins, antigens, or antibodies. The ELISA has been used as a diagnostic tool in medicine, specifically for antigen detection and the assessment of immune response, including for the determination of serum antibody concentrations for infectious diseases, like HIV, and the detection of cytokines, such as the interleukins. There are multiple forms of ELISA's including: 1) the direct ELISA employs monoclonal antibodies to detect the presence of a particular antigen in a sample; 2) the indirect ELISA is used to determine the presence of a specific antibody (e.g., HIV antibodies) in a specimen such as serum and 3) The competitive ELISA which can be used to measure various proteins using monoclonal antibodies and enzyme labeled protein.
Virtually all microbial species have at least one antigen that is unique. These antigens can be purified and used to generate specific monclonal anitbodies. Both antibodies and purified antigens provide effective and highly specific diagnostic tools.
Performing an ELISA to detect a protein involves at least one antibody with specificity for that particular protein is required. The sample with an unknown quantity of antigen or antibody is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen or antibody is immobilized, the detection antibody is added, forming a complex with the target. The detection antibody can be covalently linked to an enzyme, or can be detected by a secondary antibody which is linked to an enzyme through conjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step, the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.
Traditional ELISA involves chromogenic reporters and substrates which produce an observable color change to indicate the presence of antigen or analyte. Newer ELISA-like techniques utilize fluorogenic or electrochemiluminescent reporters to create quantifiable signals. These new reporters can have various advantages including higher sensitivities and multiplexing. Technically, newer assays of this type are not strictly ELISAs as they are not "enzyme-linked" but are, instead, linked to some non-enzymatic reporter. However, given that the general principles in these assays are largely similar, they are often grouped in the same category as ELISAs.