The plates were washed as described above,
and a colorimetric reaction was induced by the addition of
100ml chromogenic substrate (0.1 mg/ml tetramethylbenzidine
(TMB, Sigma), 100mM acetate buffer, pH 5.6,
and 1mM urea hydrogen peroxide) for different times
(from 5 to 25 min) at 37 8C.