- Tetramethylbenzidine (TMB) is a widely used chromogenic substrate for horseradish peroxidase (HRP) in biochemical and immunological assays, particularly enzyme-linked immunosorbent assays (ELISA).
- In the presence of hydrogen peroxide (H₂O₂), HRP catalyzes the oxidation of TMB, producing a soluble blue-colored product. This reaction can be quantified by measuring absorbance at 650 nm, making TMB an excellent substrate for sensitive and accurate detection of antigens, antibodies, or other biomolecules in liquid-phase assays.
- One of the defining advantages of TMB is its high sensitivity and low background, which enable reliable quantification even at very low analyte concentrations. For endpoint assays, the enzymatic reaction can be stopped by adding a strong acid, typically sulfuric or phosphoric acid. This stop solution changes the color from blue to yellow, with a stable absorbance maximum at 450 nm, allowing for reproducible spectrophotometric measurements. The strong colorimetric signal and stability of the acid-stopped product make TMB the substrate of choice in diagnostic kits, clinical assays, and research-based ELISA applications.
- In addition to ELISA, TMB is also employed in other HRP-based detection systems, such as blotting techniques and biosensors, although less commonly than insoluble substrates like DAB. Its water solubility ensures that the reaction remains homogenous in solution, allowing precise quantification rather than simple visualization. Moreover, chemiluminescent forms of TMB have been developed, further extending its sensitivity in specialized assays.
- While highly effective, TMB has some limitations and safety considerations. The substrate is light-sensitive and can degrade if not stored properly, usually requiring protection from light and storage at low temperatures. It is also moderately hazardous; TMB is considered a potential mutagen, so handling requires gloves, eye protection, and proper disposal. Overdevelopment of the color reaction can lead to signal saturation, reducing assay accuracy, which makes careful timing and standardization essential.
