Simplified Scatchard-plot assay for progesterone receptor in breast cancer: comparison with single-point and multipoint assay

Abstract

The authors describe a quantitative simplified Scatchard-plot assay for progesterone receptor with a reliability very similar to that of a six-point assay, and more reliable for detecting receptor presence than a single-point progesterone receptor assay described by Pichon and Milgrom (1977), from which it was derived. Compared to the six-point assay, this two-point assay can be used to assay smaller samples and, with positive and negative controls included, process more samples in a run. Cytosol with 4 g of protein per liter is treated separately and in triplicate with 2 concentrations (4 pmol/liter and 20 nmol/liter) of [3H]progesterone combined with hydrocortisol, 1 μmol/liter, in the absence and presence of unlabeled progesterone, 2 μmol/liter. After a 2-hr incubation at 5°C, glycerin is added to provide 300 ml/liter during a second 2-hr incubation (5°C). Bound and free progesterone are separated with charcoal. The authors tested known target and nontarget tissues for specificity and reliability, and to determine criteria for interpretation ('positive' must show K(d) <20x10-9 mol/liter with maximum binding per mg of protein >20 fmol of progesterone). Receptor was found to be stable in tissues for at least 6 mth at -60°C, but loses some activity during overnight incubation (5°C). Comparison of the two-point assay with the classical six-point assay showed complete agreement for receptor presence, with a correlation of 0.91 for maximum binding sites. Of 109 tumor tissues previously shown to possess estrogen receptor, 72 (66%) showed progesterone receptor by two-point assay. The single-point assay gave a 7% discrepancy for these samples.