These peroxidase substrates are
commonly used in enzyme-linked immunosorbent assays (ELISA),
The reaction mixture with a final volume of 1.0 mL contained a concentration of 1 mM H2O2, 10 lL of HRP isoenzyme preparation and
varying concentrations of ABTS (0.05–10 mM) or TMB (0.005–
0.5 mM) in 50 mM potassium phosphate buffer, pH 6.5.
TMB at 30 C for 180 s, respectively. Absorption curves were recorded with an adapted software program (UVPC Optional Kinetics
software, Shimadzu). The maximum reaction rate (Vmax) and the
Michaelis constant (Km) were calculated with the Sigma Plot software (Version 11.0, Systat Software Inc., USA).