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

Direct radioimmunoassay ( RIA), based on the principle of competitive inhibition for the measurement of serum progesterone using H-3 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 H-3 was used as tracer. To 12 mm x 75 mm glass tubes, 100 mu L of standard ( 250 pg to 50,000 pg/mL) and unknown samples were added, in duplicate, followed by 100 mu L of antibody and 600 mu L of tracer ( 10,000 counts per minute [ cpm]) in all of the tubes and incubated overnight at 4 degrees C. The bound and free fractions of labeled material were separated by adding 200 mu L 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).