Enzyme immunoassay for 4-hydroxy-2-(4-methylphenyl)benzothiazole.

A specific enzyme immunoassay (EIA) has been developed for 4-hydroxy-2-(4-methylphenyl)benzothiazole (KB-2714) (1), an active metabolite of 4-acetoxy-2-(4-methylphenyl)benzothiazole (KB-2683) (2) which is a promising anti-rheumatic agent. The EIA was based upon antiserum elicited against 5-(2-carboxyphenylazo)-4-hydroxy-2-(4-methylphenyl)benzothiazole (3)-bovine serum albumin conjugate and beta-galactosidase-labelled 5-amino-4-hydroxy-2-(4-methylphenyl)benzothiazole (5). The sensitivity of the EIA was significantly improved by the utilization of a bridge heterologous combination system. An appropriate dose-response curve of EIA for 4-hydroxy-2-(4-methylphenyl)benzothiazole was obtained in the range of 10 pg tube-1-20 ng tube-1. The specificity of EIA proved to be satisfactory in terms of cross-reactivities to 14 benzothiazole-related compounds including glucuronic acid and sulphuric acid conjugates. The proposed method was evaluated to be useful for the determination of 1 in urine and plasma with acceptable recovery and inter- and intra-assay precision.