DEVELOPMENT OF A TIME-RESOLVED FLUOROIMMUNOASSAY (TR-FIA) FOR TESTOSTERONE - MEASUREMENT OF SERUM TESTOSTERONE CONCENTRATIONS AFTER TESTOSTERONE TREATMENT IN THE RAINBOW-TROUT (ONCORHYNCHUS-MYKISS)

A sensitive time-resolved fluoroimmunoassay (TR-FIA) for testosterone was developed, and the assay system was used for measuring serum testo sterone concentrations in rainbow trout. Testosterone-3-(O-carboxymeth yl)oxime-bovine serum albumin (T-3-CMO-BSA) was immobilized by physica l adsorption to the wells of microtiter plates. A competitive assay us ing two antibodies was performed among T-3-CMO-BSA in the solid-phase, unknown amounts of testosterone, testosterone antibodies, and europiu m labeled secondary antibodies, followed by measurements using a time- resolved fluorometer (DELFIA system). The TR-FIA had a sensitivity of 0.075 pg/50 mu l sample (1.5 pg/ml), and the range of the assay system was between 1.5 pg/ml and 25 ng/ml. The intra- and interassay coeffic ients of variation for the testosterone TR-FIA were satisfactorily low and were between 1.62 and 6.38% and 2.96 and 8.29%, respectively. The assay system was applied to measure the serum testosterone concentrat ions after an injection of testosterone dissolved in saline, propylene glycol, or coconut oil. Among the three solvents, the coconut oil grou p showed continuously high serum testosterone levels. In contrast, the saline and propyleneglycol groups had maximum concentrations 24 hr af ter the injection, but their levels were significantly lower than that of the coconut oil group. The testosterone TR-FIA method is sensitive , repeatable, and is as accurate as conventional RIAs. It is very good for measuring serum testosterone concentrations. (C) 1997 Academic Pr ess.