Competitive Immunoassays

In competitive ELISA, the antigen in a sample competes for limited antibody binding sites with antigen conjugated to a reporter enzyme. This produces an inverse relationship between antigen concentration and substrate turnover. Competitive ELISAs typically use a single antibody to a small molecular weight antigen, generally less than 10,000 Daltons. During incubation, samples with high antigen content result in unlabeled antigen being bound in greater amounts than conjugated antigen. When chromogenic substrate is added to the assay to develop color, samples with high antigen concentration generate a lower signal than those containing low antigen concentration, yielding the inverse correlation between antigen concentration in the sample and color development in the assay. This relationship can then be used to extrapolate antigen concentration in an unknown sample from a standard curve. This type of reaction is one of the few methods possible for small molecular weight antigens, such as steroids, drugs, lipids, and peptides.

The typical format for one of our competitive ELISA kits is shown above. We use alkaline phosphatase (AP) as the reporter enzyme because of its stability, turnover number, and lack of interferences. The antibody-bound conjugate is captured using a secondary antibody that specifically binds to the primary antibody coated onto a microtiter plate.