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Method The present EIAs are described as competitive, solid-phase enzyme immunoassays. In this assay format, the antibody is captured on the solid phase and the antigen (estrogen metabolite) is labeled with the enzyme. In the test, binding of the antigen-enzyme conjugate by the antibody is inhibited by free antigen (in the standard curve, or present in the test sample). Since a restricted number of antibody binding sites are available, the enzyme activity bound to the solid phase in the presence of free antigen is lowered. When enzyme substrate is added to the washed solid phase, the enzyme product (e.g., colored dye) concentration is inversely proportional to the concentration of the free antigen. In the current assay kits, monoclonal antibodies to estrogen metabolites are captured directly to the solid phase (wells of 8 x 12 polystyrene microtiter plates). The estrogen metabolites have been conjugated to alkaline phosphatase enzyme (AP). The urinary forms of 2OHE1 and 16OHE1 are found mainly as the 3-glucuronide and or the 3,3,16-glucuronides, respectively, and require removal of the sugars before recognition by the monoclonal antibodies in the EIA kit. The estrogens are deconjugated of both glucuronic acid and sulphate by use of a mixture of ß-glucuronidase and arylsulphatase enzyme isolated from the snail Helix Pomatia (mixture is called here "Deconjugating Enzymes"). In practice, an aliquot of urine is diluted 1:20 with a buffer containing the enzymes and incubated until deconjugation is complete. The enzyme digest is then neutralized and used directly in the assay. The assay and antibodies are described more completely by Klug TL, Bradlow HL, and Sepkovic DW in Steroids 59:648-654 (1994).Comparison with other Methods The EIA kits for urinary 2OHE1 and 16OHE1 have been validated by comparing values obtained with these kits to values obtained by Gas Chromatography-Mass Spectroscopy (Adlercreutz H, Martin F, Wahlross O, Soini E. J Steroid Biochem Molec Biol 6: 247-259 (1976). With 18 urine samples from healthy premenopausal women, the linear regression correlation coefficient (r2) between the two methods was 0.94 for both 2OHE1 and 16OHE1. A more recent independent study sponsored by the National Cancer Institute (NCI) found linear regression correlation coefficients of greater than 0.98 between the GC-MS and EIA methods for both urinary metabolites in pre- and post-menopausal women {6}. The ratios of 2OHE1 to 16OHE1 obtained by the EIA method are, moreover, more consistent, and in better agreement with expected results. For example, the range of ratios for the EIA method was 1.27 to 7.9 (n=16), whereas for the GC-MS method, the range was 2.5 to 46 (n=16). The GC-MS method requires extensive urine extraction, recovery and biochemical modification techniques, with attendant errors. Furthermore, using the EIA kits, we have demonstrated 100% recovery of steroid spiked into urine samples, and recovery of steroid with sample dilution. The mean within assay variability is about 4%, the mean between assay variability is about 10%, for the experienced operator.
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Estramet 2/16 Kit
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2007. Immuna Care Corporation. |
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