Development of a sensitive microtitration plate enzyme-immunoassay for the anabolic steroid trenbolone

The development of a competitive microtitration plate enzyme-immunoassay for monitoring trenbolone application to animals is described. 'Bridge heterology' was achieved with a rabbit antibody raised against 17 beta-trenbolone-hemisuccinate-BSA and 17 alpha-trenbolone glucuronide-alkaline phosphatase as a tracer. The required 17 alpha-trenbolone glucuronide was prepared by application of trenbolone acetate to a calf following isolation from urine by three HPLC steps. The glucuronide was linked to alkaline phosphatase by the mixed anhydride procedure. The EIA was performed by coating affinity purified sheep IgG antirabbit IgG to the microtitration plate well followed by the addition of sample, tracer and hormone-specific antibody. The absolute detection limit was less than 1 pg; 50% relative binding at approximately 3 pg which is about 20 times superior to our RIA. The sample blanks of purified extracts from urine, bile, faeces, liver and muscle were similar in both methods (EIA and RIA) and much lower than required for a reliable detection of positive samples. Assay variations were always less than 15%. For the EIA, radiolabelled trenbolone, which is not commercially available, is not necessary and according to our experience the EIA is more practicable and economic.