Enzyme-linked immunosorbent assay (ELISA) kits make it easier to use ELISAs for detecting molecules or performing ligand-binding assays successfully.
Many ELISA kits use the detection enzymes horseradish peroxidase (HRP) or alkaline phosphatase (AP), along with chromogenic, fluorescent, or chemiluminescent substrates. These enzymes are conjugated to either primary or secondary antibodies.
Alternative detection strategies, such as fluorescent tags or other detectable probes, are also accepted under the umbrella term of ELISAs if they are plate-based methods, regardless of whether they use reporter enzymes or antibodies for detection. There are several formats, including direct, indirect, sandwich, and competitive ELISA.
Overview of ELISA types
The diagram below briefly summarizes the components and steps for performing the four most common ELISA types. In the most straightforward version, the direct ELISA, conjugated primary antibodies specifically bind to antigens coated in microplate wells.
Indirect ELISA follows a similar principle, but uses unlabeled primary antibodies paired with a conjugated secondary antibody.
The sandwich ELISA coats wells with primary antibodies, which in turn bind the sample antigens.
A new set of antibodies will be added, forming a sandwich on the antigens.
In the competitive ELISA, unlabeled primary antibodies are first complexed with sample antigens. These are added to wells coated with antigens. After washing, only free, unbound antibodies will bind to the coated antigens. After the addition of conjugated secondary antibodies and substrate, a signal is produced that is inversely proportional to the amount of antigens in the sample; the greater the amount of antigen in the sample, the lesser the signal. Another variant of this ELISA uses primary antibodies to coat the well. Labeled antigens will then compete with sample antigens for binding. The resulting detection signal remains inversely proportional.
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