In chemistry, TMB stands for 3,3′,5,5′-Tetramethylbenzidine, a non-carcinogenic substitute for benzidine. It's primarily used as a peroxidase substrate in enzyme-linked immunosorbent assays (ELISA).
TMB as a Peroxidase Substrate in ELISA
TMB serves as a chromogenic substrate, meaning it produces a colored product when acted upon by an enzyme, typically horseradish peroxidase (HRP). This reaction is crucial in ELISA procedures, enabling the detection and quantification of target molecules.
Reaction and Color Change
When HRP catalyzes the oxidation of TMB, it yields a soluble product. This product is typically pale blue but can change to yellow upon the addition of sulfuric acid, a common stop solution in ELISA assays. The intensity of the color is directly proportional to the amount of target molecule present.
Spectrophotometric Measurement
The colored end product is then quantified spectrophotometrically. Typically, absorbance is measured at wavelengths of 370 nm or within the range of 620-650 nm, depending on the specific assay protocol and desired sensitivity.
Key Properties and Advantages of TMB
- Non-carcinogenic: TMB offers a safer alternative to benzidine, which is a known carcinogen. This makes it a preferred choice in laboratory settings.
- High Sensitivity: TMB provides good sensitivity for detecting low concentrations of target molecules.
- Soluble Product: The soluble reaction product allows for easy spectrophotometric measurement without precipitation issues.
- Versatile: TMB is widely used in various ELISA formats and other peroxidase-based assays.
Example Use Case: ELISA for Antibody Detection
In an ELISA designed to detect antibodies against a specific antigen, TMB would be used as follows:
- Antigen is coated onto a microplate.
- The sample (e.g., serum) is added, and antibodies, if present, bind to the antigen.
- An enzyme-conjugated secondary antibody (e.g., HRP-conjugated anti-human IgG) is added, which binds to the primary antibody.
- TMB substrate is added.
- HRP catalyzes the oxidation of TMB, producing a blue colored product.
- Sulfuric acid is added to stop the reaction and change the color to yellow.
- The absorbance is measured spectrophotometrically, with higher absorbance indicating a higher concentration of antibodies against the antigen.
In summary, TMB is a valuable and widely used reagent in chemistry, particularly in immunoassays, due to its safety, sensitivity, and ease of use in generating a detectable signal.
