Electrophoretic purification of radioiodinated follicle-stimulating hormone for radioligand receptor assay and radioimmunoassay

Journal Article · Wed Oct 01 04:00:00 EDT 1986 · Endocrinology; (United States)

DOI:https://doi.org/10.1210/endo-119-4-1446· OSTI ID:7134423

Schneyer, A L; Sluss, P M; Bosukonda, D; Reichert, Jr, L E

A method is described for electrophoretic purification of (/sup 125/I)human (h) FSH after radioiodination that improves radioligand binding to FSH membrane receptors. Lactoperoxidase-iodinated hFSH was separated from reaction products by electrophoresis on 7.5% polyacrylamide tube gels (PAGE). Material eluted from 3-mm gel slices was analyzed for incorporation of /sup 125/I and binding to antibody (RIA) or receptor (RRA), and by sodium dodecyl sulfate-PAGE for protein composition. Sodium dodecyl sulfate-PAGE analysis of individual PAGE fractions demonstrated that iodinated proteins, both higher and lower in apparent mol wt than intact FSH, were separated by PAGE, but not by gel filtration chromatography (Sephadex G-25). PAGE purification of radioligand resulted in significantly greater (compared to gel filtration) RRA sensitivity and specificity. Maximum binding of PAGE-purified (/sup 125/I)hFSH to excess calf tests membrane receptors was 45%, with a specific activity of approximately 26 microCi/micrograms, as determined by the method of self-displacement. Maximum binding to excess hFSH antisera (NIH anti-hFSH 4) was 80-85%. This allowed a useful final dilution of 1:120,000, thereby facilitating development of a sensitive and specific RIA with this antiserum. These data indicate that PAGE separation of intact (/sup 125/I)hFSH from other iodinated proteins results in improved radioligand binding, assay sensitivity, and assay specificity. In addition, PAGE-purified lactoperoxidase-iodinated hFSH is suitable for use in both RIA and RRA.

• Research Organization: Albany Medical College of Union Univ., NY
• OSTI ID: 7134423
• Journal Information: Endocrinology; (United States), Journal Name: Endocrinology; (United States) Vol. 4; ISSN ENDOA
• Country of Publication: United States
• Language: English

Related Subjects

38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEAR CHEMISTRY
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62 RADIOLOGY AND NUCLEAR MEDICINE
CELL CONSTITUENTS
CELL MEMBRANES
CHEMISTRY
ELECTROPHORESIS
FSH
GONADOTROPINS
HORMONES
IMMUNOASSAY
IMMUNOLOGY
ISOTOPE APPLICATIONS
MEMBRANE PROTEINS
MEMBRANES
ORGANIC COMPOUNDS
PEPTIDE HORMONES
PITUITARY HORMONES
PROTEINS
PURIFICATION
RADIOASSAY
RADIOCHEMISTRY
RADIOIMMUNOASSAY
RADIOIMMUNOLOGY
RADIORECEPTOR ASSAY
RECEPTORS
TRACER TECHNIQUES