Competitive Assay

An affinity-purified capture antibody (typically anti-IgG) is pre-coated onto a microplate. A limited concentration of primary antibody and enzyme-linked analyte along with the sample are added simultaneously. Sample analyte and enzyme-linked analyte compete for the limited number of binding sites on the primary antibody, which is bound by the immobilized anti-IgG. Substrate is added and hydrolyzed by the enzyme, thereby producing a color product that can be measured. The amount of labeled analyte bound is inversely proportional to the amount of unlabeled analyte present in the sample (signal decreases as analyte concentration increases).