EnzymeLinkedImmunosorbentAssay

Enzyme-linked immunosorbent assay (ELISA) is a plate-based assay used to detect and quantify soluble substances such as proteins, peptides, antibodies, hormones, and antigens. The method combines antigen–antibody interactions with an enzyme-labeled detection system that yields a measurable signal, most commonly a color change detected by absorbance in a microplate reader. ELISA can be highly sensitive, specific, and adaptable to high-throughput workflows.

There are several formats used to accommodate different analytes and samples. Direct ELISA uses an enzyme-labeled primary antigen or antibody to detect the target. Indirect ELISA employs an unlabeled primary antibody and a labeled secondary antibody for detection. Sandwich ELISA requires two antibodies that flank the target, providing high specificity for larger molecules. Competitive ELISA is used for small antigens; the signal is inversely proportional to the amount of analyte in the sample because sample antigen competes with a labeled antigen for binding sites.

In a typical sandwich ELISA, a capture antibody is immobilized on a microplate well, the sample is added to bind the target antigen, a detection antibody conjugated to an enzyme is introduced, and a substrate is added to produce a colorimetric, fluorescent, or luminescent signal. Common enzymes include horseradish peroxidase and alkaline phosphatase, with substrates such as TMB or pNPP generating color changes. Readouts are compared to standard curves to determine concentrations.

ELISA is widely used in clinical diagnostics, vaccine research, basic and translational science, food safety, and quality control. Limitations include potential cross-reactivity, matrix effects, and the need for proper controls and standards. The technique was developed in the late 1970s by Eva Engvall and Peter Perlmann.