7-ALPHA-BIOTINYLATED TESTOSTERONE DERIVATIVES AS TRACERS FOR A COMPETITIVE CHEMILUMINESCENCE IMMUNOASSAY OF TESTOSTERONE IN SERUM

Ring core-biotinylated testosterone tracers were synthesized with brid ges of three different lengths connecting the biotin moiety to the ste roid core (7 alpha-C-n-Bio-T, n = 3, 6, or 11). Together with a positi on 7-specific polyclonal anti-testosterone antibody, we used the 7 alp ha-C-11-Bio-T tracer to develop a novel, labeled-hapten competitive im munoassay for total testosterone in serum. (The C-3 and C-6 tracers pr oved to be not suitable for analogous immunoassays.) Enhanced chemilum inescence signal was generated by use of a second immobilized antibody and a streptavidin-horseradish peroxidase conjugate. The measuring ra nge 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 i nterassay imprecisions are 13-16%. Accuracy determinations in mass spe ctrometry-controlled reference specimens showed a mean recovery of 95% . In addition, the assay shows low cross-reactivities, demonstrating t he favorable specificity of the combination of a ''nearly native'' tra cer with a position analog antibody. The optimized steric structure an d the long spacer arm of the biotinylated testosterone tracer make thi s chemiluminescence assay well-suited for measuring total testosterone concentration in serum.