3,3′,5,5′-Tetramethylbenzidine (TMB)

3,3′,5,5′-Tetramethylbenzidine (TMB) is a benzidine with a methyl group on each side of both amino groups. It is a chromogenic hydrogen donor for peroxidases: horseradish peroxidase (HRP), myeloperoxidase and haemoglobin oxidise it with hydrogen peroxide to coloured products.

The colour reaction. Peroxidase removes one electron from TMB to give a radical cation. The first coloured product is a blue charge-transfer complex of TMB with its two-electron oxidation product, the diimine. When the reaction is stopped with sulfuric acid, the solution turns yellow, and the yellow product is the diimine. In the aqueous assay solution, the blue product is read at about 650 nm (or at 370 nm), and the acid-stopped yellow solution at 450 nm.

ELISA and immunodetection. In enzyme-linked immunosorbent assays (ELISA), TMB detects HRP-labelled antibodies. After the plate is washed, TMB and hydrogen peroxide are added, the bound HRP develops the blue colour, and the reaction is stopped with acid and read at 450 nm. Reading at two wavelengths has been used to extend the measuring range of TMB-based ELISA. TMB is also used as an HRP substrate in staining procedures, including immunohistochemistry.

Other uses. TMB was introduced as a substitute for benzidine in the detection of blood, in which haemoglobin acts as the peroxidase. A modified TMB method is also used to measure lipid hydroperoxides.