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Enzyme-linked immunosorbent assay (ELISA) is an affinity-based method, usually carried out in multi-well plates, to determine if an antigen, often a protein or antibody, is present in a sample. Since ELISA is a common quality control and diagnostic tool in research, drug production, food production, and health care, accuracy and reproducibility is critical. The general method for ELISA begins with the immobilization of the antigen in a multi-well plate. Either the whole sample can be allowed to settle and adhere to the plate or an antibody specific to the antigen can be bound to the plate which then captures and immobilizes the antigen. The antigen is next detected either directly, when the antigen is bound by an enzyme-labeled primary antibody, or indirectly, when the bound unlabeled primary antibody is then bound by a second enzyme-labeled antibody (secondary antibody). After binding, the enzyme substrate is added, usually resulting in a color change, weak radioactivity, fluorescence, or chemiluminescence that can be detected and measured. ELISA kit development involves optimizing each step of the assay for the specific antigen to be detected and then validating the kits accuracy and reproducibility. (Credit: Brooke Anderson-White)
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