What is used as the substrate in a classical ELISA test?

In a classical ELISA (Enzyme-Linked Immunosorbent Assay), the substrate typically used is a chromogenic solution that contains a specific compound which, when acted upon by the enzyme linked to the antibody, will produce a color change. This color change is what is measured to determine the quantity of the target analyte in the sample.

In this context, hydrogen peroxide does not serve as the substrate in a classical ELISA. Instead, it is often utilized in certain enzyme reactions, particularly those involving peroxidases (such as horseradish peroxidase), where it acts as a co-substrate. However, in the classical ELISA, the enzyme's substrate is a color-producing reagent that is specifically designed for the assay, such as tetramethylbenzidine (TMB) when using horseradish peroxidase, or o-phenylenediamine (OPD), which leads to a color reaction upon oxidation.

This distinction highlights the purpose of the actual substrate: it provides the requisite component that undergoes a chemical reaction to yield the measurable result that represents the presence and quantity of the target molecule being tested.