A new spectrophotometric system of tetrabase [bis(4-dimethylaminophenyl)methane]-H₂O₂- horseradish peroxidase (HRP) was developed for the detection of HRP activity, and the reaction mechanism was discussed. The detection was based on the HRP catalyzing coloring reaction of tetrabase in pH 4.5 PBS containing KI, which behaved maximum absorption at 462 nm. The absorbance was proportional to the activity of HRP. This new tetrabase reagent is soluble and more stable than 3,3',5,5'-tetramethyl benzidine (TMB), a usually used coloring re-agent for ELISA in clinic, and could overcome the shortcomings of TMB. Under optimal conditions, the linear range for HRP detection was 2×10^－9～2.5×10^－7 g/mL with the detec-tion limit of 3×10^－10 g/mL. This system could be used for the determination of HRP labeled horse anti-human α-fetoprotein with satisfactory results. The new system is simple, rapid and sensi-tive, and has good application prospect in clinic.

Key words: spectrophotometric enzyme-linked immunosorbent assay, horseradish peroxidase, hydrogen peroxide, tetrabase, bis(4-dimethylaminophenyl)methane