TMB Substrate and Stop Solution are reagents commonly used together for colorimetric detection in enzyme-linked immunosorbent assays (ELISA) that utilize horseradish peroxidase (HRP)–conjugated detection systems. The TMB substrate contains 3,3′,5,5′-tetramethylbenzidine, which is oxidized by HRP in the presence of hydrogen peroxide to produce a blue-colored reaction product. The intensity of the blue color is proportional to the amount of HRP enzyme present and therefore reflects the quantity of the target analyte bound in the assay. To stabilize the signal and enable quantitative measurement, a stop solution—typically an acidic solution such as sulfuric or phosphoric acid—is added to terminate the enzymatic reaction, converting the blue color to a stable yellow product. This final color change allows absorbance to be measured spectrophotometrically, usually at 450 nm, using a microplate reader. TMB substrate and stop solution are widely used in ELISA assays to detect cytokines, antibodies, hormones, and other biomolecules in samples such as serum, plasma, or cell culture supernatants in research, diagnostic, and high-throughput screening applications.