The enzyme-linked immunosorbent assay (ELISA) procedure is ubiquitous in biomedical research and clinical testing. ELISA, first described by Engvall and Perlmann in 1971, is an antibody-based reaction that allows the detection and quantitation of numerous analytes with the same basic assay process.
ELISA typically uses a microplate as a solid surface on to which specific antibodies are bound allowing the capture and analysis of a seemingly infinite number of analytes. It does so by a series of incubations, reagent additions and wash-steps. During incubations specific molecules are bound to the solid substrate by interactions with the previously absorbed antibodies. Following the binding step, unbound material is removed by the washing step. After the unbound material has been removed the next reagent, usually a conjugate, is added and allowed to specifically interact. The conjugate is a chimeric molecule containing a specific antibody and an enzyme protein covalently linked. Again unbound material is removed and the next reagent, substrate added afterward. The substrate interacts with the enzymatic portion of the conjugate to produce a colored compound, which can be detected by absorbance, fluorescence or luminescence depending on the specific substrate used.
Regardless of the analyte, the same process steps are used in ELISA.
It spawned a broad range of immunoassays that utilize either wash or no-wash workflows using colorimetric, fluorometric, luminometric, TRF or TR-FRET, AlphaScreen and AlphaLISA detection technologies. Some of these latter technologies utilize low volume analysis in 1536-well plates, yet ELISA performed in 96-well microplates remains the most commonly used immunoassay technique. The ELISA technique causes formation of specific immune complexes that are easily measured using BioTek’s single or multi-mode microplate readers. The basic “building blocks” of an ELISA reaction are:
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