Sato N, Hyder S M, Chang L, Thais A, Wittliff J L
J Chromatogr. 1986 May 30;359:475-87. doi: 10.1016/0021-9673(86)80100-5.
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.
采用高效液相色谱法，基于大小（高效尺寸排阻色谱法）和表面电荷（高效离子交换色谱法）特性，从人乳腺癌中分离雌激素受体的各种异构体。评估了这些异构体与单克隆抗体相互作用的能力。所有异构体均表现出相似的免疫决定位点，但当它们与[125I]碘雌二醇-17β（IE）结合时，仅观察到放射性复合物与固定化单克隆抗体有30%的结合。然而，通过雌激素受体酶免疫测定（ER-EIA），抗体珠识别的受体质量总是显著更高。对于分级分离和未分级分离的细胞溶质均如此，这表明非配体结合形式，如雌激素受体的前体和产物，也被识别；或者配体仅选择特定的构象体；或者珠上的单克隆抗体识别与雌激素受体相关的其他蛋白质。未标记受体的离子交换分级分离显示免疫决定位点丧失。然而，尺寸排阻分级分离未显示此效应。IE结合的竞争剂己烯雌酚在尺寸排阻色谱和离子交换色谱过程中均显示ER-EIA识别的受体具有显著稳定性。对受体进行有限的胰蛋白酶处理会导致免疫决定位点丧失，而不改变配体结合位点。因此，人乳腺癌细胞溶质中雌激素受体的蛋白水解很容易导致ER-EIA低估。尽管ER-EIA识别的具有免疫决定位点的成分总是与雌激素受体的配体结合异构体一起洗脱，但我们的数据表明，具有与单克隆抗体相关表位的蛋白质浓度与类固醇配体识别的浓度不相等。我们得出结论，将ER-EIA应用于雌激素受体的临床检测显然需要进一步阐明。
