ABSTRACT
Competitive time-resolved fluoroimmunoassays (FIAs) were developed for measuring 1,3,5(10)-estratrien-3-ol-17-one glucosiduronate (estrone 3-glucuronide, E(1)3G) and 5 beta-Pregnane-3 alpha,20 alpha-diol 3-glucosiduronate (pregnanediol 3-glucuronide, Pd3G) in unextracted urine. The assays are specific, detect 0.98 ng E(1)3G/mL and 0.035 microgram Pd3G/mL, measure 102.8 +/- 2.0% of E(1)3G and 93.6 +/- 2.9% of Pd3G added, and exhibit between and within assay coefficients of variation, respectively, of 5.3% and 7.1% for E(1)3G and 6.8% and 7.8% for Pd3G. The urine matrix does not interfere with the assay. Urinary steroid glucuronide profiles measured by these FIAs conform to those of urinary steroid glucuronides and serum estradiol and progesterone measured by other established immunoassays. These FIAs afford the advantages of non-radioisotopic procedures and urine sample collection (convenience, non-invasiveness, integration of pulsatile secretion) to evaluate menstrual function in epidemiological, medical, and athletic populations.
Subject(s)
Estrogens, Conjugated (USP)/urine , Estrone/analogs & derivatives , Pregnanediol/analogs & derivatives , Antibodies, Monoclonal , Cross Reactions , Estrone/urine , Female , Fluoroimmunoassay , Humans , Immunoenzyme Techniques , Male , Menstrual Cycle/physiology , Pregnanediol/immunology , Pregnanediol/urine , Radioimmunoassay , Reference Values , Reproducibility of Results , Sensitivity and Specificity , Time FactorsABSTRACT
The applicability of bifunctional chelating agents in the labeling of proteins was analysed by using Eu-labeled antibodies as a model system. The requirements set for chelate stability are defined mainly by the system and metals used. The chelates used for in vitro applications cannot necessarily be utilized in in vivo experiments in which the competing chelating agents and metals set great demands on the inertness of the chelates against ligand changes. An isothiocyanatobenzyl derivative of diethyelenetriaminetetraacetic acid showed excellent stability in the conditions under investigation. The coupling reaction is mild and allows simple addition of metals within or after the coupling of the chelator to the protein, which makes it a good choice for the labeling of antibodies with metals like Gd3+.
