ELISA (enzyme-linked immunosorbent assay) is a plate-based immunoassay in which analyte capture is reported by an enzyme — usually HRP or alkaline phosphatase — conjugated to an antibody and converting a substrate such as TMB into a coloured product read on a spectrophotometer.
Formats, and which one a peptide needs
Four formats are standard. Direct and indirect ELISAs coat the antigen and detect it with labelled or secondary-labelled antibody. Sandwich ELISA uses a capture antibody and a detection antibody against two distinct epitopes and is the most sensitive and specific — but it requires an analyte large enough to present two epitopes simultaneously, so it is unavailable for most short peptides. Competitive ELISA solves that: labelled antigen competes with sample analyte for a limited amount of antibody, and signal falls as analyte rises. Short peptides are therefore normally quantified by competitive assay with an inverse standard curve.
Reading a kit critically means checking the stated cross-reactivity, species reactivity, and whether the calibrator is the same salt form and sequence as your sample. A four-parameter logistic fit, not a straight line, is the usual curve model.
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.