Direct Radioimmunoassay for the Measurement of Serum Progesterone using 3H as a Label

Abstract

Direct radioimmunoassay (RIA), based on the principle of competitive inhibition for the measurement of serum progesterone using 3H as label, is described. Progesterone 3‐O‐carboxymethyloxime‐ bovine serum albumin (progesterone 3‐O‐CMO‐BSA) was used as an immunogen and progesterone labeled at positions 1, 2, 6, and 7 with 3H was used as tracer. To 12 mm × 75 mm glass tubes, 100 µL of standard (250 pg to 50,000 pg/mL) and unknown samples were added, in duplicate, followed by 100 µL of antibody and 600 µL of tracer (10,000 counts per minute [cpm]) in all of the tubes and incubated overnight at 4°C. The bound and free fractions of labeled material were separated by adding 200 µL 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 the present study, a high ionic strength, along with low and neutral pH of the buffer, is utilized to release bound steroid from proteins. The sensitivity of the assay is 732 pg/mL. The recovery ranged between 94.03% to 100.96%. The inter‐assay and intra‐assay coefficients of variation ranged from 3.89% to 7.59% and from 9.96% to 12.6%, respectively. The serum progesterone values, obtained by this method, were correlated with those obtained by enzyme linked immunosorbent assay; r=0.96 (n=94).