7α-biotinylated testosterone derivatives as tracers for a competitive chemiluminescence immunoassay of testosterone in serum

Ring core-biotinylated testosterone tracers were synthesized with bridges of three different lengths connecting the biotin moiety to the steroid core (7α-C(n)-Bio-T, n = 3, 6, or 11). Together with a position 7- specific polyclonal anti-testosterone antibody, we used the 7α-C₁₁-Bio-T tracer to develop a novel, labeled-hapten competitive immunoassay for total testosterone in serum. (The C₃ and C₆ tracers proved to be not suitable for analogous immunoassays.) Enhanced chemiluminescence signal was generated by use of a second immobilized antibody and a streptavidin-horseradish peroxidase conjugate. The measuring range of the assay is 0.2-20.0 nmol/L, linearity of serial dilutions can be demonstrated, the lower detection limit is 0.125 nmol/L, and the interassay imprecisions are 13-16%. Accuracy determinations in mass spectrometry-controlled reference specimens showed a mean recovery of 95%. In addition, the assay shows low cross-reactivities, demonstrating the favorable specificity of the combination of a 'nearly native' tracer with a position analog antibody. The optimized steric structure and the long spacer arm of the biotinylated testosterone tracer make this chemiluminescence assay well-suited for measuring total testosterone concentration in serum.