Evolution of immunoreactivity of monoclonal antibodies H222 and/or D547 used in the detection of breast cancer estrogen receptors. Varying reactivity of receptor isoforms

Abstract

From 1984 to 1990, human breast cancer estrogen receptors have been measured both by a radioligand assay (RLA[ 3H]estradiol) and by an enzyme immunoassay (Abbott ER-EIA kit). The ration EIA/RLA results increased continuously from 1.04 (1984) to 1.87 (1990), and this evolution was consistent with the last trial of the E.O.R.T.C. receptor study group (Trial 1989-II, EIA/RLA = 2.5). Dilution studies of cytosols with the current ER-EIA kits showed an important parallelism defect of the standard curve, the final result of cytosols (fmol/mg protein) obtained from the upper part of the curve (between 100 and 500 fmol/ml) being 1.5 to 2 times higher than the results obtained from readings of the lower part of the standard curve (between 0 and 50 fmol/ml). Chromatographic experiments were carried out during 1986 and the measures of binding sites by RLA and of immunoreactive sites by EIA on chromatographic fractions were compared. Identical results were obtained with EIA and RLA, either on polymeric forms of the estrogen receptor, or on monomeric forms obtained after dissociation by 0.4 M KCl. The same experiments performed during 1990 showed that, in the chromatographic fractions, the concentration of immunoreactive sites was twice as large as that of ligand-binding sites, detected by tritted estratadiol. Furthermore, the detection of polymeric and monomeric receptor isoforms by monoclonal antibodies varied, and was increased by the presence of KCl (0.4 M) and/or bovine serum albumin (BSA) (1 mg/ml) in the cytosol. These findings showed that the large differences between enzyme immunoassay and ligand-binding assay results currently observed were due to differential reactivity of monoclonal antibodies for the estrogen receptor standard provided in the ER-EIA kits and for the estrogen receptor present in cytosols from human breast cancers, suggesting modifications of immunoreactivity of the monoclonal antibodies actually provided in the ER-EIA kits.