Why is TMB used in conjunction with HRP in ELISA?

HRP, or horseradish peroxidase, is commonly used in ELISA (enzyme-linked immunosorbent assay) since it can easily be conjugated to IgG and other proteins. In ELISA, TMB plays the role of a chromogenic substrate, and is also one of the most sensitive substrates for HRP.

When a TMB solution is added to HRP, HRP will reduce hydrogen peroxide and oxidize TMB, turning it from colorless to blue-green. HRP can continue in a cycle of reduction and oxidation until the reaction is stopped, which can be done by adding an acidic solution such as sulfuric acid (H2SO4) or hydrochloric acid (HCl) that turns the solution yellow.