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首页> 外文期刊>BMC Biotechnology >Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology
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Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology

机译：使用蛋白质微阵列技术筛选抗ERCC1单克隆抗体的特异性和在病理学中的应用

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Background 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. Results 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. Conclusion 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.
机译：背景技术如果用于临床目的，具有交叉反应性的抗体会产生意想不到的副作用或错误的诊断报告。 ERCC1正在探索作为基于顺铂的化学疗法的预测性诊断生物标志物。 ERCC1的高表达与基于顺铂的化学疗法的耐药性有关。 8F1是评估肺癌患者组织中ERCC1表达水平最常用的单克隆抗体之一，但已经注意到，该抗体与未知蛋白发生交叉反应。结果通过使用高密度蛋白质微阵列芯片技术，我们发现8F1不仅与其真实靶标ERCC1反应，而且还与无关的核膜蛋白PCYT1A发生交叉反应。交叉反应性是由于这两个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测试表明PCYT1A主要定位在核膜上。在这项研究中，我们还发现在某些肺癌患者组织样本中，PCYT1A基因表达水平明显高于ERCC1基因表达水平。为了开发用于ERCC1 IHC分析的最佳单克隆抗体，产生了18种单克隆抗体，并针对我们的蛋白质微阵列芯片筛选了其中的6种。两个克隆在蛋白质微阵列芯片测试中显示出很高的单特异性，并且都适用于IHC应用。结论总之，由于8F1克隆与PCYT1A蛋白具有交叉反应性，因此不适合用于ERCC1 IHC分析。两种新产生的单克隆抗体4F9和2E12对ERCC1蛋白表现出超特异性，并且在IHC分析中表现出色。

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