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Enzyme-Linked ImmunoSorbent Assay, or ELISA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. Thus in the case of fluorescence ELISA, when light is shone upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be measured.
Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen 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 is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation. 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. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates with much higher sensitivity.
Enzyme ImmunoAssay (EIA) is a synonym for the ELISA.
Additional recommended knowledge
Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations (such as with the HIV test or West Nile Virus) and also for detecting the presence of antigen. It has also found applications in the food industry in detecting potential food allergens such as milk, peanuts, walnuts, almonds, and eggs. The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity. In an ELISA test, a person's serum is diluted 400-fold and applied to a plate to which HIV antigens have been attached. If antibodies to HIV are present in the serum, they may bind to these HIV antigens. The plate is then washed to remove all other components of the serum. A specially prepared "secondary antibody" — an antibody that binds to human antibodies — is then applied to the plate, followed by another wash. This secondary antibody is chemically linked in advance to an enzyme. Thus the plate will contain enzyme in proportion to the amount of secondary antibody bound to the plate. A substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or fluorescence. ELISA results are reported as a number; the most controversial aspect of this test is determining the "cut-off" point between a positive and negative result.
It is also used in testing cats for the FIV disease.
Prior to the development of the EIA/ELISA, the only option for conducting an immunoassay was radioimmunoassay, a technique using radioactively-labeled antigens or antibodies. In radioimmunoassay, the radioactivity provides the signal which indicates whether a specific antigen or antibody is present in the sample. Radioimmunoassay was first described in a paper by Rosalyn Sussman Yalow and Solomon Berson published in 1960.
Because radioactivity poses a health threat, a safer alternative was sought. A suitable alternative to radioimmunoassay would substitute a non-radioactive signal in place of the radioactive signal. When certain enzymes (such as peroxidase) react with appropriate substrates (such as ABTS or 3,3’,5,5’-Tetramethylbenzidine), they can result in changes in color, which can be used as a signal. However, the signal has to be associated with the presence of antibody or antigen, which is why the enzyme has to be linked to an appropriate antibody. This linking process was independently developed by Stratis Avrameas and G.B. Pierce. Since it is necessary to remove any unbound antibody or antigen by washing, the antibody or antigen has to be fixed to the surface of the container, i.e. the immunosorbent has to be prepared. A technique to accomplish this was published by Wide and Porath in 1966
In 1971, Peter Perlmann and Eva Engvall at Stockholm University in Sweden, as well as Anton Schuurs and Bauke van Weemen in The Netherlands, independently published papers which synthesized this knowledge into methods to perform EIA/ELISA
The steps of the general, "indirect," ELISA for determining serum antibody concentrations are:
The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules. A major disadvantage of the indirect ELISA is that the method of antigen immobilization is non-specific; any proteins in the sample will stick to the microtiter plate well, so small concentrations of analyte in serum must compete with other serum proteins when binding to the well surface. The sandwich ELISA provides a solution to this problem.
ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical. Two or three times the standard deviation is often used to distinguish positive and negative samples. In quantitative ELISA, the optical density or fluorescent units of the sample is interpolated into a standard curve, which is typically a serial dilution of the target.
A less-common variant of this technique, called "sandwich" ELISA, is used to detect sample antigen. The steps are as follows:
The image to the right includes the use of a secondary antibody conjugated to an enzyme, though technically this is not necessary if the capture antibody is conjugated to an enzyme. However, use of a secondary-antibody conjugate avoids the expensive process of creating enzyme-linked antibodies for every antigen one might want to detect. By using an enzyme-linked antibody that binds the Fc region of other antibodies, this same enzyme-linked antibody can be used in a variety of situations. The major advantage of a sandwich ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present. Without the first layer of "capture" antibody, any proteins in the sample (including serum proteins) may competitively adsorb to the plate surface, lowering the quantity of antigen immobilized.
A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different than the first two examples:
For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal.
(Note that some competitive ELISA kits include enzyme-linked antigen rather than enzyme-linked antibody. The labeled antigen competes for primary antibody binding sites with your sample antigen (unlabeled). The more antigen in the sample, the less labeled antigen is retained in the well and the weaker the signal.)
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "ELISA". A list of authors is available in Wikipedia.|