The 3,3',5,5'-Tetramethylbenzidine (TMB) liquid substrate system is widely used in ELISA (Enzyme-Linked Immunosorbent Assay) for detecting the presence of antigens or antibodies. It serves as a chromogenic substrate that produces a blue color when oxidized by the enzyme horseradish peroxidase (HRP), which is commonly conjugated to secondary antibodies in ELISA protocols. The reaction is stopped by adding an acid, turning the solution yellow, and the intensity of the color is measured spectrophotometrically to quantify the target analyte. TMB is preferred due to its high sensitivity, low background noise, and compatibility with both visual and instrumental detection methods. It is extensively applied in clinical diagnostics, biomedical research, and pharmaceutical development for detecting biomarkers, infectious agents, and immune responses.