The Detection of Relaxin in Porcine, Ovine and Human Plasma by Radioimmunoassay

A radioimmunoassay for porcine relaxin is described based on the inhibition of the reaction between I-labeled porcine relaxin and rabbit antiserum to porcine relaxin. The time course of the binding of labeled porcine relaxin to antiserum was determined by chromatoelectrophoresis. The sensitivity of the assay is greater than 4.0 ng of relaxin/ml of plasma. Immunoactivity in plasma from a late pregnant sheep and pig was identical to the standard porcine relaxin. Inhibitions obtained with plasmas from an early pregnant sheep, a ram, boar and normal man differed slightly from that of the standard porcine relaxin. On the other hand, definite partial crossreactions were shown by both pig and sheep plasma after ovariectomy and no inhibition was observed by plasma from a sheep and human after both ovariectomy and hysterectomy. {Endocrinology 91: 1113, 1972) T N 1926, Hisaw (1) described a peptide hor•*mone isolated from the corpora lutea of sow ovaries which produced a marked relaxation of the pelvic ligaments in guinea pigs. Bioassays were subsequently developed based upon the biological activities of relaxin, Hall (2). Current bioassay methods are impractical for measurements of relaxin in multiple samples taken in a wide variety of physiological conditions. Hemagglutination-inhibition has been used for relaxin measurements in saline extracts of ovaries from several species and for cross-reactivity studies by Cohen (3) and McClintock and Zarrow (4). The application of this technique to the estimation of plasma relaxin activity has not been reported. The relaxin preparation used for this study (NIH-R-P1) was extracted from the ovaries of pregnant sows and therefore represents ovarian relaxin as produced during pregnancy. The Uterine Relaxing Factor (URF) content of this preparation is not known. URF has not yet been identified as a separate moiety from relaxin, Griss, Keck, Englehorn and Tuppy (5), isolated from pig ovaries. However, in addition to the possible heterogeneity of ovarian relaxin, plasma may also contain relaxins produced by the decidua, endometrium and placenta, according to Dallenbach and Dallenbach-Hellweg (6). Received January 4, 1972. Address for correspondence: Dr. Gillian D. Bryant, Department of Anatomy & Reproductive Biology, 1960 East-West Road, Honolulu, Hawaii 96822. A sensitive and precise radioimmunoassay has been developed for ovarian relaxin and preliminary results have been obtained in plasma from pig, sheep and man. Evidence of a uterine source of relaxin is shown and discussed. Materials and Methods Buffers. Sodium salts were used in the preparation of buffers. Diluent buffer refers to a solution 0.5 mg/ml of bovine gamma globulin, Fraction II (Nutritional Biochemicals Corporation) in 0.05M-barbitone buffer pH 8.6 with 0.5 mg/ml iodoacetamide (Nutritional Biochemicals Corporation). Horse serum was obtained in liquid form from Grand Island Biological Company. Porcine relaxin. A preparation of porcine relaxin (NIH-R-Pl) was used as a standard and for the preparation of I-labeled porcine relaxin. Polyacrylamide gel electrophoresis at pH 7.6 and 9.6 showed a single band but further studies on the physicochemical homogeneity are in progress. i'-labeled porcine relaxin. This was prepared weekly by the method of Hunter and Greenwood (7) except that 0.5 mCi obtained from Cambridge Nuclear, Mass., U.S.A. was reacted with the hormone (10 Mg in 0.02 ml) and 30 M-g of chloramine-T. The 1 3 Ilabeled porcine relaxin was separated from the unreacted iodide on Sephadex G-25 (1.5 g) previously equilibrated with 1 ml of 0.05M-barbitone buffer, pH 8.6, containing 20 mg of bovine gamma globulin. Eluates (15 X 0.75 ml) in the 0.05M-barbitone buffer were collected into tubes containing 0.1 ml of diluent. The fraction containing the highest number of counts in the protein peak was repurified by passage through Sephadex G-50 (2.5 g) previously