Effect of OPD and enzyme concentration: optimized assay.
By systematically varying the concentrations of OPD and
H2O2, we obtained the highest absorbance value for the
chromophore at 435 nm by using 100 mmol/L OPD and 20
mmol/L H2O2. These absorbance values for a 10- to 20-mm
incubation are about eightfold those obtained by other investigators
(2-6) using the OPD assay for HRP. The conditions
giving those values are those of our optimized assay.