Secondary antibodies for ELISA are linked to enzymes. Detection of secondary antibodies that are bound to primary antibodies occurs by an enzyme-substrate reaction. In this kit, the secondary antibody is linked to horseradish peroxidase (HRP). In the presence of hydrogen peroxide (H2O2), HRP catalyzes the oxidation of the chromogenic substrate 3,3’,5,5’-tetramethylbenzidene (TMB). This oxidation of TMB by HRP forms a blue product. Note: TMB is light sensitive, and the assay results should be determined 5–10 minutes after the substrate is added to the wells. If the microplate strips sit longer, nonspecific color may develop. Color that develops after the 5-minute incubation should not be considered in the assay results. After 20–30 minutes, the blue color may begin to fade as TMB precipitates out of solution.