ELISA

ELISA, the enzyme-linked immunosorbent assay, is an immunoassay run on a microplate. Capture of the analyte is signalled by an enzyme — typically HRP or alkaline phosphatase — coupled to an antibody; the enzyme turns a substrate such as TMB into a coloured product that a spectrophotometer reads.

Choosing the right format for a peptide

There are four established formats. In direct and indirect ELISA the antigen is coated onto the plate and detected with a labelled antibody or a labelled secondary. The sandwich format pairs a capture antibody with a detection antibody aimed at two separate epitopes; it offers the best sensitivity and specificity, but only works when the analyte is big enough to display two epitopes at once, which rules it out for most short peptides. The competitive format gets around this: labelled antigen and the analyte in the sample compete for a limited quantity of antibody, so the signal drops as analyte concentration climbs. For that reason, short peptides are usually measured by competitive assay with an inverse calibration curve.

Evaluating a kit properly means reviewing the declared cross-reactivity and species reactivity, and confirming that the calibrator matches your sample in sequence and salt form. Curves are normally fitted with a four-parameter logistic model rather than a straight line.

Related terms and material

RIA · immunogen · blocking peptide. Reference material: assay kits. Further reading: ELISA kits for peptide research and how ELISA and RIA kits quantify peptides.