Use of Calf Uterine Cytosol Estrogen Receptor Coupled to Class Beads as a Stable Internal Control for Estradiol Receptor Assay in Human Breast Cancer

Abstract

Calf uterine tissue was homogenized in six volumes (w/v) of 10 mM Tris-HCl buffer of pH 7.4, containing 1 mM monothioglycerol and 10% glycerol (TG) and centrifuged at 800 ± g for ten minutes. The supernatant was centrifuged at 105,000 ± g for 60 minutes to obtain the cytosol fraction. Sand-blasted uniferm glass beads (6 mm diameter) were regcted with 10% gamma-aminopropyl triethoxysilane in toulene at 60°C for seven hours to generate aminopropyl derivatives. The derivatized beads were activated with 2.5% glutaraldehyde and incubated overnight with the calf uterine cytosol receptor at 4°C. After incubation, the beads were washed with Tris-HCl buffer and stored at -20°C. The cytosol receptor coupled heads were incubated for 18 hours with and without 100 fold excss of diethylstilbesterol. The beads were suspended in scintillation fluid and counted for ten minutes. The binding sites and dissociation constant (K_D) of the calf uterine cytosol receptor coupled to beads were 7.2 fmol/bead or 144 fm/mg protein and 4.3 ± 10⁻⁹ M, respectively. There wae little loss of the cytosol estrogen receptor coupled to glass beads up to a period of three months. Calf uterine cytosol estrogen receptor coupled to beads provided a stable internal control to monitor the inter-assay variations due to the reagents, radioisotopes, and method used in measurement of E₂ receptor in human breast cancer cytosol fraction in pre-and post-menopausal women. The interassay precision was 4.6% (P < 0.05). The mean concentration of E₂ and receptors was 19 fm per mg protein in pre-menopausal women; however, no significant difference was observed in the proportion of E₂ receptor positive tumors in pre-and post-menopausal women.