A radio-immunoassay of 19-nortestosterone using Celite column chromatography

In order to measure plasma or serum concentrations of 19-nortestosterone (nandrolone), a radio-immunoassay has been developed using C7-tritium labelled nandrolone and antisera raised against nandrolone 3-(O-carboxymethyl)oxime-albumin. Because these antisera displayed considerable cross-reactions towards testosterone, 5 alpha-dihydrotestosterone and 5 alpha-dihydronandrolone, it proved to be necessary to separate nandrolone from these cross-reacting steroids prior to the radio-immunoassay. Using Celite/ethylene glycol columns a complete separation could be achieved between nandrolone and these cross-reacting steroids. The assay procedure involved extraction with ethyl acetate, chromatography on Celite/ethylene glycol columns, overnight incubation of samples and standards with antiserum and labelled compound, bound/free separation with dextran-coated charcoal and counting of the supernatant in a liquid scintillation counter. Recoveries for the total assay procedure (expressed as fractions) were between 0.85 and 1.01. The assay displayed a sensitivity of 0.7 nmol/l (at 50% binding) or 0.2 nmol/l (at 80% binding), while the within-assay and from day to day coefficients of variation were 9.8% (n = 114) and 12.9% (n = 13) respectively for duplicate measurements.