Dittadi R, Meo S, Amoroso B, Gion M
Centro Regionale Specializzato per lo Studio degli Indicatori Biochimici di Tumore, Ospedale Civile, Campo SS. Giovanni e Paolo, Venezia, Italy.
Cancer Res. 1997 Mar 15;57(6):1066-72.
Estrogen receptors (ER) are routinely measured in tissue extracts from breast cancer using a radioligand binding assay (RBA) and an enzyme immunoassay (EIA). Although good correlation was found between the two methods, they are expected to measure, at least in part, different ER amounts in individual samples, because the RBA should detect unfilled ER only, whereas EIA should recognize both unfilled receptors and those filled by endogenous estrogens. The purpose of the present investigation was to evaluate if ER-EIA mainly detects ER filled by endogenous estrogens when using an estrogen-free buffer to dilute cytosol samples. Indeed, the commercially available EIA assay kit (ER-EIA; Abbott) is equipped with a sample dilution buffer containing a high concentration of 17beta-estradiol which should allow for the saturation of all the ERs. ER was measured in 57 cytosol samples from primary breast cancer with RBA and ER-EIA. In the latter case, samples were diluted using both the estradiol-rich dilution buffer of the kit and an estrogen-free low salt phosphate buffer. RBA and ER-EIA showed tightly correlated results. However, ER-EIA detected higher ER levels than RBA in the majority of cases. Results obtained by low salt ER-EIA were also correlated to both RBA and ER-EIA, showing, however, lower ER concentrations. ER levels measured by ER-EIA were not significantly different from the sum of ER concentrations found by RBA and low salt ER-EIA. These findings suggest that ER-EIA detects ER only in the conformational status that is achieved after saturation by estrogens. These findings were confirmed by sedimentation shift experiments, which showed that the monoclonal antibody D547 used in the kit binds ER in the occupied form only. This leads to the conclusion that ER-EIA detects functioning (in terms of binding with estradiol) ERs. From the present investigation, we suggest that it is possible and probably worthwhile to optimize the EIA method by using different buffers to measure: (a) the total number of ERs capable of binding estradiol; (b) the ER filled by endogenous estrogens; and (c) by difference, the unfilled ER concentrations.
雌激素受体（ER）通常通过放射性配体结合测定法（RBA）和酶免疫测定法（EIA）在乳腺癌组织提取物中进行检测。虽然这两种方法之间存在良好的相关性，但预计它们至少在一定程度上测量的是单个样本中不同的ER量，因为RBA应该只检测未结合的ER，而EIA应该识别未结合的受体和那些被内源性雌激素占据的受体。本研究的目的是评估当使用无雌激素缓冲液稀释胞质溶胶样本时，ER-EIA是否主要检测被内源性雌激素占据的ER。实际上，市售的EIA检测试剂盒（ER-EIA；雅培公司）配备了一种含有高浓度17β-雌二醇的样本稀释缓冲液，该缓冲液应能使所有的ER饱和。使用RBA和ER-EIA对57例原发性乳腺癌的胞质溶胶样本进行了ER检测。在后一种情况下，样本分别使用试剂盒中富含雌二醇的稀释缓冲液和无雌激素的低盐磷酸盐缓冲液进行稀释。RBA和ER-EIA显示出紧密相关的结果。然而，在大多数情况下，ER-EIA检测到的ER水平高于RBA。低盐ER-EIA获得的结果也与RBA和ER-EIA相关，但显示出较低的ER浓度。ER-EIA测量的ER水平与RBA和低盐ER-EIA发现的ER浓度之和没有显著差异。这些发现表明，ER-EIA仅检测在雌激素饱和后达到的构象状态下的ER。沉降迁移实验证实了这些发现，该实验表明试剂盒中使用的单克隆抗体D547仅结合被占据形式的ER。这得出结论，ER-EIA检测有功能的（就与雌二醇结合而言）ER。从本研究中，我们建议通过使用不同的缓冲液来测量以下各项，优化EIA方法是可行的，而且可能是值得的：（a）能够结合雌二醇的ER总数；（b）被内源性雌激素占据的ER；以及（c）通过差值计算，未结合的ER浓度。
