- Enzyme-linked immunosorbent assay (ELISA) is based on an older and very similar technique called enzyme immunoassay (EIA). These immunological assays are biochemical in nature and are used to detect and quantify the presence of an antibody or an antigen in a sample. ELISA assays are applied in various fields of biotechnology and molecular biology and are being used as a diagnostic tool in medicine, as well as a quality control assay in various industries.
ELISA assays allow for the detection of an unknown amount of antigen which is bound to a surface. A specific antibody is applied on the antigen bound to the surface so that it can bind to the antigen. The antibody is linked to an enzyme as reporter agent that produces a detectable signal. Often these reporters produce a light of specific wave-length (fluorescence) if expose to UV-light. The amount of antigen in the sample can be determined by comparing the light intensity to a standard of know concentration.
There are several types of ELISA assays that are based on the same main principle but vary slightly in their approach. Main distinctions are direct and indirect ELISAs. Thereby the ELISA is used to detect the presence of antigens that are recognized by an antibody (direct method) or it can be used to test for antibodies that recognize an antigen (indirect method). Further distinctions include the sandwich ELISA, which measures the amount of antigen between two layers of antibodies. Sandwich assays are restricted because the antigens to be measured must contain at least two antigenic sites, since at least two antibodies act in the sandwich.
In so-called competitive ELISAs an unlabelled primary antibody is coated onto the wells of a 96 well microtiter plate. This primary antibody is then incubated with unlabelled standards and samples with unknown protein content. Conjugated antigen or enzyme-linked antibody is then added which will bind to the primary antibody wherever its binding sites are not already occupied. The more unlabelled antigens are to be found in the sample or standard, the lower the amount of conjugated antigen bound.