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Clinical Chemistry 21: 1725-1730, 1975;
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Clinical Chemistry, Vol 21, 1725-1730, Copyright © 1975 by the American Association for Clinical Chemistry

Simplified Scatchard-Plot Assay for Estrogen Receptor in Human Breast Tumor

Roy B. Johnson Jr. 1, Robert M. Nakamura 1, and Rose Mary Libby 1

1 Scripps Clinic and Research Foundation and Department of Pathology, Hospital of Scripps Clinic, 476 Prospect St., La Jolla, Calif. 92037.

In this two-point Scatchard-plot assay, with which a test of competitive inhibition is combined, the sample is mechanically homogenized in a buffer containing dithiothreitol, ultracentrifuged to obtain a fat-free cytosol, the protein content of which is then adjusted, and free and bound labeled estradiol are separated with dextrancoated charcoal after overnight incubation. We tested the method for precision and reliability by assaying such cytosols from pregnant rabbit uteri before and after dilution with kidney cytosol, and by assaying several other target and nontarget animal and human tissues. The Scatchard plot data were more reliable than tests for percent inhibition of binding by a competitor (diethylstilbestrol). For a tumor tissue to be judged positive it must bind at least 8 fmol of estradiol per milligram of protein and have a Kd of 0.1 to 5 x 10-10 mol/liter. Some nontarget tissues showed less than 70% inhibition by 104-fold concentrations (over labeled estradiol) of inhibitor. Of 19 breast-tumor specimens, seven were found to be positive.


Key Words: test of competitive inhibition • in hibition of binding by a competitor • cancer • estrogenbinding protein • assessing value of endocrine therapy

Submitted on June 2, 1975
Accepted on August 8, 1975




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Copyright © 1975 by the American Association for Clinical Chemistry.