A radioimmunoassay for the regulatory allylic steroid, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP).
The allylic steroid, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP), found in gonadal and brain tissues by radiotracer and chemical methods, had been shown to play a role in gametogenesis, gonadotropin secretion and brain excitability. Since no simple assay was available, a radioimmunoassay for 3 alpha HP was developed using [3H]3 alpha HP and an antiserum raised against 3 alpha HP-20-CMO conjugated to bovine serum albumin. The specificity of the assay for the 3 alpha allylic configuration of 3 alpha HP was confirmed by examining 32 other steroids; cross-reaction with steroids containing different configurations (including metabolites of 3 alpha HP such as progesterone) was less than 0.9%. A Scatchard plot indicated a Ka of 1.56 X 10(9) M-1. Inter- and intra-assay coefficients of variation were 13.1 and 4.5%, respectively. The sensitivity of the assay was 6 pg and the 50% intercept of the standard curve was approx. 123 pg. The measurement by RIA of 3 alpha HP from standard solutions and HPLC purified tissue extracts was confirmed qualitatively and quantitatively by GC/MS methods. The RIA method was employed to determine 3 alpha HP levels in cultured Sertoli cells and in serum of intact and ovariectomized adult rats. Although for most uses, chromatography would not be necessary, two possible methods are presented to enable the separation of 3 alpha HP from other interfering steroids prior to RIA.
Wiebe
J P
JP
Department of Zoology, University of Western Ontario, London, Canada.
Barr
K J
KJ
Buckingham
K D
KD
D004092:20-alpha-Dihydroprogesterone / Q000031:analogs & derivatives* / Q000032:analysis / Q000276:immunology; D000818:Animals; D000918:Antibody Specificity; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D005069:Evaluation Studies as Topic; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D008297:Male; D010052:Ovariectomy; D011374:Progesterone / Q000032:analysis; D011817:Rabbits; D011863:Radioimmunoassay / Q000379:methods*; D051381:Rats; D011919:Rats, Inbred Strains; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012708:Sertoli Cells / Q000737:chemistry
D004092:20-alpha-Dihydroprogesterone; C052610:3-hydroxypregn-4-en-20-one; D011374:Progesterone