Measuring the nandrolone threshold ratio by enzyme-linked immunosorbent assay for 5α-estrane-3β,17α-diol

Abstract The international threshold for nandrolone in equine urine is reached when the ratio of 5α-estrane-3β,17α-diol to 5(10)-estrene-3β,17α-diol exceeds one. When this ratio is exceeded, the sample is positive for administration of a nandrolone preparation. The present method for measuring the ratio requires hydrolysis of the urine sample followed by clean-up procedures, then analysis of the derivatized residue by gas chromatography-mass spectrometry (GCMS). A quicker measurement of the ratio for screening purposes is desirable. A hybridoma that secretes monoclonal anti-5α-estrane-3β,17α-diol antibody has been developed. Using inhibition enzyme-linked immunosorbent assay (ELISA), this monoclonal antibody detects 5α-estrane-3β,17α-diol at 1 ng ml −1 and is far more sensitive than a rabbit polyclonal antiserum. The monoclonal antibody is specific to 5α-estrane-3β,17α-diol, 5α-estrane-3β-ol-17-one and 5α-estrane-3β-ol-17-carboxymethyloxime. Slight cross-reactivity with other structurally related steroids is observed at concentrations higher than 200 ng ml −1 . Work is in progress to raise a monoclonal antibody against 5(10)-estrene-3β,17α-diol. The two ELISAs should provide a rapid screening test to measure the nandrolone threshold ratio. Detailed GC-MS analysis would then only be required for confirmation.