Comparison of O-phenylenediamine dihydrochloride (OPD) and Tetramethylbenzidine (TMB) as horseradish peroxidase substrate systems for use in ELISA

Different chromogenic substrate mixtures are used with different enzyme labels for developing ELISA. O-phenylenediamine dihydrochloride (OPD) and tetramethylbenzidine (TMB) are commonly employed horseradish peroxidase substrate systems. Tetramethylbenzidine and OPD were compared for sensitivity in Pasteurella multocida serum antibody titre determination ELISA. The antibody titres were calculated in end point indirect ELISA using these two substrate systems. OPD and TMB substrates were found comparable when antibody titres with both substrates were analysed statistically. Therefore, TMB could be a better substrate than OPD due to continuous risk of carcinogenic property of OPD.