Application

o-Phenylenediamine dihydrochloride has been used as a substrate for horseradish-peroxidase-conjugated secondary antibody in enzyme-linked immunosorbent assay (ELISA).

General description

o-Phenylenediamine dihydrochloride (OPD) is a chromogenic substrate that can be used in enzyme-linked immunosorbent assay (ELISA) procedures as it utilizes horseradish peroxidase conjugates. The substrate produces a soluble end product that is orange-brown and can be read spectrophotometrically at 450 nm. The OPD reaction may be stopped with 3N HCl or 3M H2SO4 and read at 492 nm.

o-Phenylenediamine (o-PD) is a colorless and nonfluorescent molecule.

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.