Interaction of estrogen receptor isoforms with immobilized monoclonal antibodies.
Interaction of estrogen receptor isoforms with immobilized monoclonal antibodies.
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雌激素受体亚型与固定化单克隆抗体的相互作用。
DOI：
10.1016/0021-9673(86)80100-5
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Wittliff,JL
中科院分区：
文献类型：
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作者：
Sato,N;Hyder,SM;Chang,L;Thais,A;Wittliff,JL
采用高效液相色谱法分离人乳腺癌雌激素受体的各种同种异构体，基于大小（高效尺寸排除色谱法）和表面电荷（高效离子交换色谱法）的性质。评估了这些同工型与单克隆抗体相互作用的能力。所有同种异构体都显示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30%的放射性复合物与固定化单克隆抗体的结合。然而，通过雌激素受体-酶免疫测定（ER-EIA），抗体头识别的受体质量总是显著更高。这对于分离和非分离的细胞质都是正确的，这表明(1)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(2)配体只选择一个特定的构象；或(3)头部的单克隆抗体识别与雌激素受体相关的其他蛋白。未标记受体的离子交换分离显示免疫决定位点的缺失。然而，粒径排除分馏没有显示出这种效果。己烯雌酚作为IE结合的竞争对手，在大小排斥和离子交换色谱中都表现出明显的ER-EIA识别受体的稳定性。有限的胰蛋白酶处理受体导致免疫决定位点的丧失而不改变配体结合位点。因此，人乳腺癌细胞质中雌激素受体的蛋白水解很容易导致ER-EIA低估。尽管雌激素受体的配体结合异构体总是能洗脱具有雌激素受体识别的免疫决定位点的成分，但我们的数据表明，具有与单克隆抗体相关的表位的蛋白浓度与类固醇配体识别的蛋白浓度是不相等的。我们得出结论，ER-EIA在雌激素受体临床检测中的应用显然需要进一步澄清。
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-17β (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 (1) non-ligand binding forms, such as precursors and products of the estrogen receptor, were also recognized; or (2) the ligand was only selecting for a particular conformer(s); or (3) 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.