Clinical Chemistry
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Clinical Chemistry 37: 2069-2075, 1991;
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Clinical Chemistry, Vol 37, 2069-2075, Copyright © 1991 by American Association for Clinical Chemistry

Ultrasensitive, specific, two-antibody immunoradiometric assay that detects free alpha subunits of glycoprotein hormones in blood of nonpregnant humans

S Grover, J Griffin and WD Odell
Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City 84132.

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.


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Follicle-Stimulating Hormone and Its {{alpha}} and {beta} Subunits in Rainbow Trout (Oncorhynchus mykiss): Purification, Characterization, Development of Specific Radioimmunoassays, and Their Seasonal Plasma and Pituitary Concentrations in Females
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Copyright © 1991 by the American Association for Clinical Chemistry.