Development and evaluation of a competitive time-resolved immunofluorometric assay for the estrogen-regulated protein pS2.
We have developed a competitive assay to measure the estrogen-regulated protein pS2. A monoclonal pS2 antibody (mAb) and a biotinylated pS2 peptide are used, with time-resolved fluorometry as a detection technique. The assay has a detection limit of 16 ng/mL and is precise (within-run and day-to-day Cvs 3-12%). We used this assay to determine steroid hormone activity of six steroids in cell culture, both in terms of time course and dose response. pS2 concentrations in the tissue culture supernatant of the BT-474 breast carcinoma cell line were significantly higher when estradiol was the stimulating steroid. There was a significant time course and dose response observed for estradiol, but not for the other steroids. The availability of a sensitive, reliable, and convenient method for quantifying pS2 will allow for many research applications including the screening of natural and synthetic compounds for putative estrogenic activity.
Rosenberg Zand
R S
RS
Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
Jenkins
D J
DJ
Diamandis
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EP
D000450:Aldosterone / Q000494:pharmacology; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D001710:Biotin / Q000737:chemistry; D001943:Breast Neoplasms; D002138:Calibration; D002624:Chemistry, Clinical / Q000379:methods*; D003907:Dexamethasone / Q000494:pharmacology; D013196:Dihydrotestosterone / Q000494:pharmacology; D004306:Dose-Response Relationship, Immunologic; D029721:Drosophila Proteins; D004958:Estradiol / Q000494:pharmacology*; D005069:Evaluation Studies as Topic; D005455:Fluorescent Antibody Technique; D005470:Fluorometry / Q000379:methods; D005938:Glucocorticoids / Q000494:pharmacology; D039001:Integrin alpha Chains; D016023:Integrins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D008969:Molecular Sequence Data; D009277:Nandrolone / Q000031:analogs & derivatives / Q000494:pharmacology; D009644:Norgestrel / Q000494:pharmacology; D045167:Progesterone Congeners / Q000494:pharmacology; D012680:Sensitivity and Specificity; D045165:Testosterone Congeners / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism
D000911:Antibodies, Monoclonal; D029721:Drosophila Proteins; D005938:Glucocorticoids; D039001:Integrin alpha Chains; D016023:Integrins; D045167:Progesterone Congeners; D045165:Testosterone Congeners; C082188:if protein, Drosophila; D013196:Dihydrotestosterone; D009644:Norgestrel; D000450:Aldosterone; D004958:Estradiol; D009277:Nandrolone; D001710:Biotin; D003907:Dexamethasone; C100075:mibolerone