Direct radioimmunoassay for the measurement of serum testosterone using 3H as label

Direct radioimmunoassay (RIA), based on the principle of competitive inhibition for the measurement of serum testosterone, using 3H as label, is described. Testosterone 3-O-carboxymethyloxime-bovine serum albumin (testosterone 3-O-CMO-BSA) was used as an immunogen and testosterone, labeled at positions 1, 2, 6, and 7 with 3H was used as tracer. To 12 x 75 mm glass tubes 100 microL of standard (250 to 10,000 pg/mL) and unknown samples were added in duplicate, followed by 100 microL of antibody and 600 microL of tracer (10,000 counts per minute [cpm]) in all the tubes and incubated overnight at 4 degrees C. The bound and free fraction of labeled were separated by adding 200 microL of charcoal followed by centrifugation. The bound radioactivity was measured in the supernatant by using a scintillation fluid containing 2,5-diphenyloxazole (PPO, primary scintillator) and p-bis[2-(5-phenyloxazolyl)]-benzene (POPOP, secondary scintillator). In this new strategy, high ionic strength and low pH of the buffer are utilized to release bound steroid from proteins. The sensitivity of the assay is 270 pg/mL. The analytical recovery ranged from 100.24% and 108.94%. The inter-assay and intra-assay coefficients of variation ranged from 3.38% to 9.56% and 5.69% to 9.84%, respectively. The serum testosterone values obtained by this method were correlated with those obtained by solid phase radioimmunoassay: r = 0.91 (n=34).