- o-Phenylenediamine dihydrochloride (OPD) is a classical chromogenic substrate used with horseradish peroxidase (HRP) in enzyme-linked immunosorbent assays (ELISA) and other immunoassays for the colorimetric detection of antigens and antibodies.
- In the presence of hydrogen peroxide (H₂O₂), HRP catalyzes the oxidation of OPD to produce a soluble orange–yellow product. The resulting color can be measured spectrophotometrically, typically at 450–492 nm after stopping the reaction with acid, allowing sensitive and quantitative detection of analytes in biological samples.
- One of the key features of OPD is its high sensitivity and solubility, which make it well-suited for solution-based assays such as ELISA. Unlike insoluble chromogens such as DAB or AEC that are designed for tissue staining, OPD ensures a homogenous reaction in liquid phase, resulting in precise absorbance readings. Its reaction product is stable for a reasonable period after acid stopping, which supports accurate endpoint measurements and facilitates batch processing in high-throughput laboratory workflows.
- OPD has historically been a widely used substrate in diagnostic assays, especially before the rise in popularity of tetramethylbenzidine (TMB). While both OPD and TMB provide high sensitivity, TMB is generally preferred today due to its stronger signal, better stability, and improved safety profile. Nonetheless, OPD remains relevant in certain research and diagnostic contexts, particularly where assay systems are validated with OPD or where the orange color provides a distinct advantage for visual monitoring.
- Despite its utility, OPD carries important safety concerns. It is a suspected mutagen and carcinogen, requiring strict handling precautions including the use of gloves, lab coats, eye protection, and appropriate waste disposal. OPD is also light-sensitive and should be stored in dark containers under cool conditions to preserve stability. In practice, laboratories that continue to use OPD often prepare substrate solutions fresh before use to avoid degradation and variability.
