Simplified Scatchard-plot assay for estrogen receptor in human breast tumor.

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 dextran-coated 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 non-target tissues showed less than 70% inhibition by 10(4)-fold concentrations (over labeled estradiol) of inhibitor. Of 19 breast-tumor specimens, seven were found to be positive.