Simple radioimmunoassay of five unconjugated ovarian steroids in a single sample of serum or plasma.

A simplified radioimmunoasay procedure is described for determination of free ovarian steroids in plasma or serum. Ether extracts were chromatographed on a Sephadex LH-20 column equilibrated with benzene-methanol (85:15 by vol) and estrone, estradiol, estriol, progesterone, and llcw-hydroxyprogesterone were resolved from each other. With relatively specific antibodies, all five hormones were specifically assayed directly after the single chromatographic procedure. Procedures for additional purification are described when specific antibodies are not available. A mechanized chromatographic apparatus was used to purify 12 serum extracts simultaneously, permitting up to 120 determinations per day by a single worker. Assay reliability criteria were satisfactory for all assay systems, and steroid values obtained from subjects in different physiological states agreed with previously reported concentrations.