o-Phenylenediamine dihydrochloride

o-Phenylenediamine dihydrochloride has been used as a substrate for horse radish peroxidase in detecting:

• human immunoglobulin
• CTLA4Ig in serum samples
• albumin in culture medium

o-Phenylenediamine dihydrochloride is a binucleophile and its condensation with α,β-unsaturated carbonyl compounds and aldehydes yields benzimidazoles.

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.

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.