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