This greatly reduces background noise and consequently these assays are most popular for determining antibody titers in samples.ĭisadvantages of the indirect ELISA include a longer protocol with more opportunities for errors and potential of cross-reactivity with the secondary antibody.ĭeveloped in 1977 6, as its name suggests, the sandwich ELISA sandwiches the antigen between antibodies. However, if the assay is used for the detection of sample antibodies, purified target antigen is coated onto the plates, with the primary antibody coming from the sample. Where total sample antigens are bound to the plates, like the direct ELISA, background noise remains an issue. It also negates the need for target-specific conjugated detection antibodies/antigens as the conjugated secondary antibody need only be species specific for the primary antibody. This enables amplification of the test signal, overcoming the limitation of the direct ELISA. Originally developed in 1978 5 for the detection of human serum albumin, the indirect ELISA, or iELISA, works in a very similar way to the direct ELISA except for the addition of a secondary antibody step. It also requires conjugated detection antibodies/antigens to be created for each target required. The absence of a secondary antibody step means there is no signal amplification, reducing assay sensitivity. However, as the adsorption step is non-specific, background noise may be high. Following this, a detection substrate is used to produce a measurable color change that can be quantified in a plate reader.Īs this assay has few steps, it is quicker and offers less opportunity for the introduction of errors than the other ELISA methods. A conjugated detection antibody or antigen specific for the target is then applied to the wells. With a direct ELISA, the antigens or antibodies in a sample are adsorbed directly to the test plate in a non-specific manner. The images below (Figure 1) illustrate detection of antigens however, the same principle applies for antibody detection essentially with the roles of the antigen and primary antibody reversed. There are now four main types of ELISA, direct, indirect, sandwich and competitive. They sought an immunoassay method able to detect the presence of antigens or antibodies to replace the radioimmunoassay, which employed potentially hazardous radioactively labeled antigens or antibodies, and thus devised an enzyme-based alternative. The ELISA was originally conceptualized, independently, in 1971 by Eva Engvall and Peter Perlman 3 at Stockholm University in Sweden, and Anton Schuurs and Bauke van Weemen 4 in the Netherlands. A detection step involving a conjugated antigen, or more often antibody, is then employed to enable successful binding to be detected and quantified, most often by colorimetric detection. There are several different assay formats, but all rely on binding of either the target itself or an antibody/antigen able to capture the target to the surface of the plate. It is, however, important to consider that some sample types may include inhibitory factors, such as buffer components that share similar antigenic epitopes 1 or factors like proteases 2 that may damage the target or detection components, that may interfere with the assay’s performance. Serum, plasma, cell culture supernatants, cell lysates, saliva, tissue lysates and urine are all common sample types used for these assays, but most liquid sample types could be used in theory. Typically, ELISA assays are performed in 96-well plates, a format that makes them amenable to screening many samples at once. Like other immunoassays, they rely on binding of antibodies to their targets to facilitate detection. ELISA test results, what does a positive ELISA test tell you?Īpplications of ELISAs and the LAM ELISA testĪn ELISA is an immunological assay commonly used to measure antibodies or antigens, including proteins or glycoproteins, in biological samples.
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