Electrophoretic purification of radioiodinated follicle-stimulating hormone for radioligand receptor assay and radioimmunoassay.
A method is described for electrophoretic purification of [125I]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 125I 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 [125I]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 [125I]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.
D000818:Animals; D002417:Cattle; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D005640:Follicle Stimulating Hormone / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D007106:Immune Sera / Q000276:immunology; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D008297:Male; D008970:Molecular Weight; D011863:Radioimmunoassay; D011869:Radioligand Assay; D011962:Receptors, FSH / Q000378:metabolism; D013737:Testis / Q000378:metabolism
D007106:Immune Sera; D007457:Iodine Radioisotopes; D011962:Receptors, FSH; D005640:Follicle Stimulating Hormone