A radioimmunoassay for the measurement of plasma progesterone is described. The 21-hemisuccinate of 11-desoxycortisol was coupled to human serum albumin using carbodiimide, and the conjugate was used to immunize 2 ewes. The antiserum obtained after 8 months of immunization from one of the ewes was used in the assay procedure. Incubation at 4 C overnight of this diluted antiserum was carried out in the presence of l,2-3 H-17-hydroxyprogesterone. Separation of free from bound hormone was achieved by a dextran-coated charcoal suspension. One ml of plasma or serum, with tritiated progesterone added for recovery, was extracted with ether, then chromatographed on a microcolumn of Celite. Recovery of 3 Hprogesterone after extraction and chromatography was 84.2+4.8% (SD). The sensitivity varied between 10 and 25 pg of progesterone. Recovery experiments of progesterone added to water and plasma confirmed accuracy. Concentration of progesterone in plasma from men averaged 233 ±64 (SD) pg/ml and from postmenopausal women 180 ±63 (SD) pg/ml. During the follicular and midluteal phases of the menstrual cycle, the levels of plasma progesterone were, respectively: 545 ±103 pg/ml (mean±SD) and 8561+4661 pg/ml. During pregnancy, the plasma level of progesterone varied with the stages of pregnancy, being 48.4 ±18 ng/ml (mean ±SD) at 16-18 weeks of gestation, 98.0 ±28 at 28-30 weeks and 178.5 ±48 at term. (J Clin Endocr 32: 619, 1971)

Summary. It is not practical to quantitate gonadotropin in the blood of normal men and women by utilizing bioassays. We have developed a method for sensitive, precise, and specific radioimmunoassay of luteinizing hormone (LH) in human serum or plasma. Antisera were developed against human chorionic gonadotropin, and one of these was selected for extensive cross-reaction with human LH. Highly purified LH was radioiodinated by the method of Greenwood, Hunter, and Glover. Separation of antibody-bound from free LH-131I was accomplished by a double antibody technique. Dose-response curves for the purified LH, an impure urinary LH preparation, pituitary powder, and LH in plasma were all identical. Immunoassay and bioassay of impure urinary and pituitary gonadotropin preparations in terms of a common standard resulted in an index of discrimination of close to unity. LH levels in plasma from 32 adult men and 30 women outside the midcycle ranged from 0.6 to 3.2 mfAg per ml (1 m/Ag of our laboratory LH standard is equivalent to 8 mU of the Second International Reference Preparation of Human Menopausal Gonadotropin). Levels were remarkably constant in men from day to day and in women except at midcycle, when a sharp peak occurred lasting less than 24 hours. In all women studied who had a midcycle LH peak, mean plasma LH levels during the follicular phase of the menstrual cycle were higher than mean values obtained during the luteal phase. Prepubertal children had detectable plasma LH, and mean values were only slightly less than in adults. Plasma from castrate men or women or postmenopausal women contained 4.5 to 10.5 mug per ml. Clomiphene treatment of four men resulted in a doubling of plasma LH in 5 days. Introduction Wide, Roos, and Gemzell (1), using an immunoassay based on hemagglutination inhibition, first reported that human luteinizing hormone (HLH) cross-reacts with human chorionic gonadotropin (HCG) for binding to antibodies directed against HCG. In 1964 Paul and Odell (2) first developed a radioimmunoassay for HCG.

Plasma levels of FSH, LH, progesterone (P), 17-hydroxy-progesterone (17-OHP), and estradiol-173 (E 2 ) were measured daily by radioimmunoassay in eleven menstrual cycles from eleven normally menstruating women. The following criteria were used as indirect evidence for a normal ovulatory cycle: 1) mid-cycle LH peak, 2) luteal phase of 12-16 days duration, and 3) plasma progesterone above 5 ng/ml 5-8 days after the LH peak. Nine of eleven cycles studied were considered normal. A mid-cycle peak of E 2 and 17-OHP was observed in every normal cycle studied.In one of the normal cycles, two peaks of plasma E 2 occurred respectively on days 9 and 22. However, 17-OHP levels were low on day 9 and elevated on day 22. A gonadotropin surge occurred on day 22 followed by a normal luteal phase. The other two abnormal cycles studied either showed low levels of E 2 and 17-OHP or a peak of only one of the two steroids. The data suggest that in women, there is a relationship between the steroids 17-OHP and E 2 , and the mid-cycle surge of gonadotropins. (/ Clin Endocr 34: 312, 1972) T HE ADVENT of radioimmunoassay (1) has permitted quantitation of polypeptide hormones in small volumes of biological materials and has made frequent sampling feasible (2-4). Due to this breakthrough in methodology, the pattern of plasma immunoreactive FSH and LH during the normal menstrual cycle has been established (5-18). More recently, the principle of radioimmunoassay was applied to the measurement of steroid hormones in plasma (19-33). Using this and other similar techniques, various investigators have studied the relationship between the mid-cycle surge of gonadotropins and plasma levels of progestins and estrogens (24,34-43). These studies have improved our knowledge concerning mechanisms involved in the mid-cycle ovulatory surge of gonadotropins in women. In an attempt to tackle this question, we have studied daily plasma levels of the gonadotropins LH and FSH and of the steroid hormones progesterone (P), 17-hydroxyprogesterone (17-OHP), and estradiol-17(3 (E 2 ) during eleven Received August 6, 1971. menstrual cycles. The results of this study suggest that, in women, 17-hydroxyprogesterone and estradiol-17(3 are both related to the mid-cycle surge of gonadotropins. Materials and MethodsSubjects. Eleven women in apparent good health on no medications and with histories of regular menstrual cycles were studied. The clinical data on these subjects are summarized in Table 1. The follicular phase (pre-LH peak duration) was denned as the time interval between the first day of menses and the LH peak. The luteal phase (post-LH peak duration) was the interval between the LH peak and the first day of the following menses. The following criteria were used to define a normal ovulatory cycle (38,44):1) mid-cycle LH peak; 2) luteal phase duration between 12 and 16 days; and 3) plasma progesterone levels above 5 ng/ml 5-8 days after the LH peak.Method. Plasma FSH (17), LH (5), progesterone (23), 17-hydroxyprogesterone (24), and estradiol-17(3 (25) were...

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