Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology.
Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology.
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DOI：
10.1186/1472-6750-12-88
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发表时间：
2012-11-21
影响因子：
3.5
通讯作者：
He WW
中科院分区：
文献类型：
--
作者：
Ma D;Baruch D;Shu Y;Yuan K;Sun Z;Ma K;Hoang T;Fu W;Min L;Lan ZS;Wang F;Mull L;He WW
如果用于临床目的，具有交叉反应性的抗体可能会产生意想不到的副作用或错误的诊断报告。ERCC1正被探索作为顺铂化疗的预测性诊断生物标志物。ERCC1的高表达与顺铂化疗的耐药性有关8F1是用于评估肺癌患者组织中ERCC1表达水平的最常用的单克隆抗体之一，但是已经注意到该抗体与未知蛋白质交叉反应。利用高密度蛋白质芯片技术，我们发现8F1不仅与其真正的靶点ERCC1反应，而且还与一个无关的核膜蛋白PCYT1A交叉反应。交叉反应性是由于这两种不相关的蛋白质上存在共同的表位。与ERCC1的亚细胞定位相似，IHC检测表明PCYT1A主要定位于核膜上。在本研究中，我们还发现在一定人群的肺癌患者组织样本中，PCYT1A基因的表达水平明显高于ERCC1基因的表达水平。为了开发用于ERCC1 IHC分析的最佳单克隆抗体，产生了18个单克隆抗体，并对我们的蛋白质微阵列芯片筛选其中的6个。两个克隆在蛋白芯片检测中显示出高度的单特异性，并且都适用于IHC应用。总之，由于8F1克隆与PCYT1A蛋白具有交叉反应性，因此其不适用于ERCC1 IHC检测。两个新产生的单克隆抗体，4F9和2E12，表现出对ERCC1蛋白的超特异性和上级的IHC分析性能。
An antibody with cross-reactivity can create unexpected side effects or false diagnostic reports if used for clinical purposes. ERCC1 is being explored as a predictive diagnostic biomarker for cisplatin-based chemotherapy. High ERCC1 expression is linked to drug resistance on cisplatin-based chemotherapy. 8F1 is one of the most commonly used monoclonal antibodies for evaluating ERCC1 expression levels in lung cancer patient tissues, but it has been noted that this antibody cross-reacts with an unknown protein. By using a high density protein microarray chip technology, we discovered that 8F1 not only reacts with its authentic target, ERCC1, but also cross-reacts with an unrelated nuclear membrane protein, PCYT1A. The cross-reactivity is due to a common epitope presented on these two unrelated proteins. Similar to the subcellular localization of ERCC1, IHC tests demonstrated that PCYT1A is localized mainly on nuclear membrane. In this study, we also discovered that the PCYT1A gene expression level is significantly higher than the ERCC1 gene expression level in a certain population of lung cancer patient tissue samples. To develop the best monoclonal antibody for ERCC1 IHC analysis, 18 monoclonal antibodies were generated and 6 of them were screened against our protein microarray chip. Two clones showed high mono-specificity on the protein microarray chip test and both worked for the IHC application. In summary, the 8F1 clone is not suitable for ERCC1 IHC assay due to its cross-reactivity with PCYT1A protein. Two newly generated monoclonal antibodies, 4F9 and 2E12, demonstrated ultra-specificity against ERCC1 protein and superior performance for IHC analyses.
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影响因子：
6.4
作者：
Costa Lima, Lucianne Maia;de Souza, Ludmilla Regina;Carvalho, Heloisa de Andrade
通讯作者：
Carvalho, Heloisa de Andrade
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通讯作者：
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通讯作者：
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影响因子：
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作者：
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