Typically performed in multi-well microtiter plates, ELISAs are a molecular biology assay commonly used for the detection and quantification of diverse molecules, including peptides, proteins, and antibodies. Such assays can detect molecules of interest at the pg/mL level and are critical for both basic research and disease research application needs.
The fundamental molecular components of an ELISA typically include the use of antibody conjugated to an enzyme, an immobilized molecule(s) of interest, and a detection substrate. A critical aspect that determines the success and quality of data obtained from an ELISA is dependent on the affinity and specificity of antibody-antigen interactions. Antigen-antibody interactions are influenced by numerous factors, including pH, temperature, and ionic strength.
ELISAs that use direct detection methods require an immobilized antigen that is bound directly to the surface of an assay plate or indirectly by a capture antibody, followed by an antigen-specific primary antibody conjugated to an enzyme, and the detection substrate. The more commonly used indirect detection format incorporates both an unconjugated primary antibody, followed by a conjugated secondary antibody that is specific to the detection of the primary antibody. Indirect detection benefits from increased immunoreactivity with the target antigen as the conjugated enzyme element is only present on the secondary antibody. In addition to direct and indirect detection methods, capture or “sandwich” assays use an additional antigen-capturing antibody that is first attached to the microplate surface, followed by the use of both a primary and an enzyme-conjugated secondary antibody, similar to the indirect method previously described.