3,3′,5,5′-Tetramethylbenzidine or TMB is a chromogenic substrate used in staining procedures in

immunohistochemistry Immunohistochemistry is a form of immunostaining. It involves the process of selectively identifying antigens in cells and tissue, by exploiting the principle of Antibody, antibodies binding specifically to antigens in biological tissues. Alber ...

as well as being a visualising

reagent In chemistry, a reagent ( ) or analytical reagent is a substance or compound added to a system to cause a chemical reaction, or test if one occurs. The terms ''reactant'' and ''reagent'' are often used interchangeably, but reactant specifies a ...

used in enzyme-linked immunosorbent assays (

ELISA The enzyme-linked immunosorbent assay (ELISA) (, ) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay is a solid-phase type of enzyme immunoassay (EIA) to detect the presence of ...

). TMB is a white solid that forms a pale blue-green liquid in solution with

ethyl acetate Ethyl acetate commonly abbreviated EtOAc, ETAC or EA) is the organic compound with the formula , simplified to . This flammable, colorless liquid has a characteristic sweet smell (similar to pear drops) and is used in glues, nail polish removers, ...

. TMB is degraded by sunlight and by

fluorescent lights A fluorescent lamp, or fluorescent tube, is a low-pressure mercury-vapor gas-discharge lamp that uses fluorescence to produce visible light. An electric current in the gas excites mercury vapor, to produce ultraviolet and make a phosphor ...

. Used to detect

hematuria Hematuria or haematuria is defined as the presence of blood or red blood cells in the urine. "Gross hematuria" occurs when urine appears red, brown, or tea-colored due to the presence of blood. Hematuria may also be subtle and only detectable with ...

as it turns blue in contact with

hemoglobin Hemoglobin (haemoglobin, Hb or Hgb) is a protein containing iron that facilitates the transportation of oxygen in red blood cells. Almost all vertebrates contain hemoglobin, with the sole exception of the fish family Channichthyidae. Hemoglobin ...

Enzymatic assay

TMB can act as a hydrogen donor for the reduction of

hydrogen peroxide Hydrogen peroxide is a chemical compound with the formula . In its pure form, it is a very pale blue liquid that is slightly more viscosity, viscous than Properties of water, water. It is used as an oxidizer, bleaching agent, and antiseptic, usua ...

to water by

peroxidase Peroxidases or peroxide reductases ( EC numberbr>1.11.1.x are a large group of enzymes which play a role in various biological processes. They are named after the fact that they commonly break up peroxides, and should not be confused with other ...

enzymes such as

horseradish peroxidase The enzyme horseradish peroxidase (HRP), found in the roots of horseradish, is used extensively in biochemistry applications. It is a metalloenzyme with many isoforms, of which the most studied type is C. It catalyzes the oxidation of various or ...

. The resulting one-electron oxidation product is a

diimine Diimines are organic compounds containing two imine (RCH=NR') groups. Common derivatives are 1,2-diimines and 1,3-diimines. These compounds are used as ligands, but they are also precursors to other organic compounds. Preparation Diimines are ...

-diamine complex, which causes the solution to take on a blue colour, and this colour change can be read on a spectrophotometer at the wavelengths of 370 and 650 nm. The reaction can be halted by addition of

acid An acid is a molecule or ion capable of either donating a proton (i.e. Hydron, hydrogen cation, H+), known as a Brønsted–Lowry acid–base theory, Brønsted–Lowry acid, or forming a covalent bond with an electron pair, known as a Lewis ...

or another stop reagent. Using

sulfuric acid Sulfuric acid (American spelling and the preferred IUPAC name) or sulphuric acid (English in the Commonwealth of Nations, Commonwealth spelling), known in antiquity as oil of vitriol, is a mineral acid composed of the elements sulfur, oxygen, ...

turns TMB yellow, with a peak absorbance of 450 nm. The amount of converted TMB may be indexed by the amount of 450 nm light it absorbs.

Material safety

TMB should be kept out of direct sunlight as it is photosensitive. It is not known if TMB is carcinogenic and the evidence is contradictory: TMB is not mutagenic by the

Ames test The Ames test is a widely employed method that uses bacteria to test whether a given chemical can cause mutations in the DNA of the test organism. More formally, it is a bioassay, biological assay to assess the mutagenic potential of chemical com ...

, and did not induce formation of tumors in a single-arm study of 24 rats. On that evidence, it has been used as a replacement for carcinogenic compounds such as

benzidine Benzidine (trivial name), also called 1,1'-biphenyl-4,4'-diamine (systematic name), is an organic compound with the chemical formula, formula (C6H4NH2)2. It is an aromatic amine. It is a component of a test for cyanide. Related derivatives are ...

and o-phenylenediamine.

References

{{DEFAULTSORT:Tetramethylbenzidine, 3,3,5,5- Biphenyls Anilines