Application

o-Phenylenediamine Free Base is a peroxidase substrate suitable for use in ELISA procedures. The substrate produces a soluble end product that is orange-brown in color and can be read spectrophotometrically at 450 nm. The OPD reaction may be stopped with 3 N HCl or 3 M H₂SO₄ and read at 492 nm.

o-Phenylenediamine has been used to detect transforming growth factor (TGF-β) and to measure anti-cTfRMAb-ScFv fusion protein using enzyme linked immunosorbent assay (ELISA).

Biochem/physiol Actions

o-Phenylenediamine (o-PD) is a potent substrate for spectrophotometric horseradish peroxidase (HRP)-mediated enzyme-linked immunosorbent assay (ELISA), which gives 2,3-diaminophenazine (DAP) as a product of o-PD oxidation.

Reconstitution

Dissolve one tablet in 0.05 M phosphate-citrate buffer, pH 5.0, to the desired concentration (typically an OPD concentration of 0.4 mg/ml is used). Add 40 µl of fresh 30% hydrogen peroxide per 100 ml of substrate buffer solution, immediately prior to use. Tablets are individually packaged in foil packets.