Pitfalls in the Dextran-coated charcoal assay of estrogen receptors in breast cancer tissue.

This study investigated the influence of the degree of concentration of breast tumor cytosols on the apparent estrogen receptor content as measured by the Dextran-charcoal assay. It was found that the dilution of cytosols to 1-2 mg protein/ml frequently but not always causes highly underestimated receptor concentrations. This could not be explained by the protein loss through adsorption to the charcoal. The effect was also studied in the presence of gelatin, sodium molybdate or with limited trypsinization of the incubation mixture. Addition of 1 mg/ml gelatin in the Dextran-charcoal suspension was very useful in most cases in preventing dilution induced losses in receptor sites. Both trypsinization and addition of sodium molybdate produced increases in receptor concentrations that were not as susceptible to inactivation through dilution of the cytosol. These data suggest that the observed high variability in the dilution induced receptor losses can be explained by receptor heterogeneity: some receptor form(s) are either readily absorbed to or "stripped" by the charcoal particles. As a conclusion we recommend that in order to optimize the estrogen receptor assay as regards both binding sites and affinities the cytosol concentrations should be maintained as high as possible and a protein expander be included in the Dextran-charcoal suspension. Though sodium molybdate frequently gives considerable increases in estrogen binding sites it occasionally has an opposite effect. For this reason we hesitate to recommend its use in routine assays of estrogen receptors.