Interaction of estrogen receptor isoforms with immobilized monoclonal antibodies.
High-performance liquid chromatography was performed to separate the various isoforms of estrogen receptor from human breast cancer, based on size (high-performance size-exclusion chromatography) and surface charge (high-performance ion-exchange chromatography) properties. The ability of these isoforms to interact with the monoclonal antibodies was assessed. All isoforms exhibited similar immunodeterminant sites, but when they are bound to [125I]iodoestradiol-17 beta (IE), only 30% binding of the radioactive complex to the immobilized monoclonal antibodies was observed. However, the mass of the receptor recognized by the antibody bead, via the estrogen receptor-enzyme immunoassay (ER-EIA), was always significantly higher. This was true for both fractionated and non-fractionated cytosols, suggesting that non-ligand binding forms, such as precursors and products of the estrogen receptor, were also recognized; or the ligand was only selecting for a particular conformer(s); or the monoclonal antibody on the bead recognized other proteins associated with estrogen receptor. Ion-exchange fractionation of unlabeled receptor showed loss of immunodeterminant sites. However, size-exclusion fractionation did not show this effect. Diethylstilbestrol, a competitor of IE binding, showed marked stability of receptor recognized by ER-EIA during both size-exclusion and ion-exchange chromatography. Limited trypsin treatment of the receptor caused the loss of immunodeterminant sites without altering the ligand binding sites. Thus, proteolysis of estrogen receptors in cytosols of human breast cancer could easily lead to underestimation by ER-EIA. Although the components with immunodeterminant sites recognized by ER-EIA were always eluted with the ligand-binding isoforms of the estrogen receptor, our data suggest that the concentration of the protein having the epitope associated with the monoclonal antibody is unequal to that recognized by the steroid ligand. We conclude that application of ER-EIA to clinical assays of estrogen receptors clearly needs further clarification.
D000911:Antibodies, Monoclonal; D001943:Breast Neoplasms / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000032:analysis; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D011960:Receptors, Estrogen / Q000032:analysis*; D014357:Trypsin
D000911:Antibodies, Monoclonal; D011960:Receptors, Estrogen; D014357:Trypsin