title: Ultrasensitive, specific, two-antibody immunoradiometric assay that detects free alpha subunits of glycoprotein hormones in blood of nonpregnant humans.
abstract: This noncompetitive, sensitive, immunoradiometric assay of the free alpha subunit of human pituitary glycoprotein hormones is based on two monoclonal antibodies and an avidin-biotin separation system. The affinity of the first antibody, mouse anti-alpha subunit covalently conjugated to biotin, is 3.8 x 10(11) L/mol. The second antibody, radiolabeled with 125I, has an affinity of 5.4 x 10(11) L/mol. A polystyrene ball coated with avidin serves as the separation system. Tests of "purified" immunochemical-grade intact human glycoprotein hormones yielded cross-reactions of approximately 2% in the assay. Sephadex G-100 column chromatography showed that this "cross-reaction" was caused by contamination of the various hormone preparations with free alpha subunit. When the intact glycoprotein hormones were further purified with specific anti-alpha monoclonal antibody, their reaction in the alpha subunit assay was undetectable (less than 0.01%). Interassay CV averaged 3.5%, and intra-assay CV averaged 7.5% at low concentrations of subunit. The detection limit of the assay (0.01 micrograms/L) is adequate to detect free alpha subunit in the blood of normal humans. Mean (SD) concentrations of free alpha subunit in normal humans were as follows: eugonadal men = 437 (35) ng/L; postmenopausal women = 1231 (40) ng/L; eugonadal women, follicular phase = 1061 (40) ng/L; eugonadal luteal phase = 780 (45) ng/L.
authors.affiliation: Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City 84132.
authors.forename: S
authors.initials: S
authors.lastname: Grover
authors.forename: J
authors.initials: J
authors.lastname: Griffin
authors.forename: W D
authors.initials: WD
authors.lastname: Odell
mesh_terms: D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal; D001360:Avidin; D001710:Biotin; D005260:Female; D015292:Glycoprotein Hormones, alpha Subunit / Q000097:blood*; D006801:Humans; D015592:Immunoradiometric Assay / Q000379:methods* / Q000706:statistics & numerical data; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D012016:Reference Values
chemical_list: D000911:Antibodies, Monoclonal; D015292:Glycoprotein Hormones, alpha Subunit; D001360:Avidin; D001710:Biotin