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Validation of the use of C‐2/C‐16α estrogen metabolites as markers for the action of chemopreventive agents in the prevention of breast cancer
Author(s) -
Bradlow H. Leon,
Sepkovic Daniel W.,
Klug Thomas,
Osborne Michael P.
Publication year - 1993
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.240531149
Subject(s) - chemistry , hydrolysate , urine , monoclonal antibody , estrogen , breast cancer , chromatography , immunoassay , conjugate , reagent , antibody , cancer , pharmacology , medicine , biochemistry , immunology , hydrolysis , mathematical analysis , mathematics
Studies from this laboratory have demonstrated that negative modulation of the C‐2/C‐16α ratio of estradiol metabolites serves as a marker of the action of oncogenes and carcinogens which increase tumorigenicity, while positive modulation of this ratio measures the preventive effects of chemotherapeutic and chemopreventive agents on tumors and tumor cells. In order to facilitate human studies on chemopreventive agents and facilitate the measurement of this ratio, we have validated an ELISA assay using monoclonal antibodies developed by Immunocare, Inc., coated to 96‐well plates. Urine samples (10λ) were diluted in buffer and hydrolyzed with mixed glucuronidase and sulfatase to cleave the conjugates. Aliquots of the hydrolysate were added to ELISA plates coated with the C‐2 and C‐16α antibodies respectively and the appropriate labeled antigens were added. After incubation the plates were washed, the color reagent added, and the plates read kinetically to determine the amount of compound present. A standard curve is run on each plate along with high and low standards. All samples were run in triplicate and the mean values determined. The ratios were computed automatically by the reader. Blind comparisons of duplicate urine samples showed a mean r value of 97%. Mean intra‐assay variability was under 8% and inter‐assay variability was under 10%. Studies involving diet modification and the differences between breast cancer patients and controls are underway.