3,3′,5,5′-Tetramethylbenzidine (TMB) is a noncarcinogenic substitute (Ames test negative) for benzidine suitable as peroxidase substrate for use in ELISA procedures. The substrate produces a soluble end product that is pale blue in color and can be read spectrophotometrically at 370 or 620-650 nm. The TMB reaction may be stopped with 2 M H₂SO₄ (resulting in a yellow color), and read at 450 nm. A sensitive and specific reagent for the detection of blood, assay of hemoglobin, assay of peroxidases.

3,3′,5,5′-Tetramethylbenzidine has been used:

• as a supplement in Man-Rodosa-Sharpe (MRS) agar plate, to stains the lactobacilli colonies blue in the presence of O2
• as a substrate solution for ELISA (enzyme-linked immunosorbent assay)
• in myeloperoxidase activity assay to quantify neutrophil

3,3′,5,5′-Tetramethylbenzidine/TMB can be used as a chromogen to increase the progression of product obtained in peroxidase reaction. In food and environmental decontamination procedures, TMB can be used in in situ free available chlorine (FAC) monitoring of chlorite-based sanitizers.

Bottomless glass bottle. Contents are inside inserted fused cone.