A Radioimmunoassay for\u27plasma Progesterone

A radioimmunoassay for progesterone was developed which uses one micro-column chromatography for purification of hexane extracts of plasma. Progesterone conjugated at the C-3 position by an oxime with bovine serum albumin was used to produce antisera in rabbits. Plasma volumes of 0.05 ml from women in the mid-luteal phase and 0.5 ml from women at other times in the menstrual cycle and from men are adequate for duplicate determinations of progesterone. Plasma samples were mixed with known amounts of 1,2-3H-progesterone and extracted with hexane. The extracts were applied directly to Al2O3 microcolumns. The columns were washed with 0.28% ethanol in hexane and the progesterone eluted with 0.45% ethanol in hexane. The dried eluates were incubated at room temperature for two hours with a 1:4,500 dilution of antiserum in buffer. Saturated (NH4)2SO4 was used to separate free from bound progesterone. The tritium in the supernatant was counted. The accuracy and the precision of the method were satisfactory. The sensitivity was 25 pg per sample. The results obtained by this method in the analysis of plasma from normal men and women were in good agreement with those reported by other investigators using different methods. One technician can measure 22 plasma samples in duplicate plus 2 parallel standards in 1 1 2 days