Successful translation of findings derived from preclinical studies into effective therapies is critical in biomedical research. Lack of robustness and reproducibility of the preclinical data, due to insufficient number of repeats, inadequate cell-based and mouse models contribute to the poor success rate. Antibodies are widely used in preclinical research, notably to determine the expression of potential therapeutic targets in tissues of interest, including tumors, but also to identify disease and/or treatment response biomarkers. We sought to determine whether the current antibody characterization standards in preclinical research are sufficient to ensure reliability of the data found in peer-reviewed publications. To address this issue, we used detection of the protein c-FLIP, a major factor of resistance to apoptosis, as a proof of concept. Accurate detection of endogenous c-FLIP levels in the preclinical settings is imperative since it is considered as a potential theranostic biomarker. Several sources of c-FLIP antibodies validated by their manufacturer and recommended for western blotting were therefore rigorously tested. We found a wide divergence in immune recognition properties. While these antibodies have been used in many publications, our results show that several of them failed to detect endogenous c-FLIP protein by Western blotting. Our results suggest that antibody validation standards are inadequate, and that systematic use of genetic knockdowns and/or knockouts to establish proof of specificity is critical, even for antibodies previously used in the scientific literature. Because antibodies are fundamental tools in both preclinical and clinical research, ensuring their specificity is crucial.

译文

成功地将临床前研究的发现转化为有效的疗法在生物医学研究中至关重要。由于重复次数不足,基于细胞的和小鼠模型不足,缺乏临床前数据的稳健性和可重复性导致成功率低。抗体广泛用于临床前研究,特别是用于确定感兴趣组织 (包括肿瘤) 中潜在治疗靶标的表达,还用于鉴定疾病和/或治疗反应生物标志物。我们试图确定临床前研究中当前的抗体表征标准是否足以确保同行评审出版物中发现的数据的可靠性。为了解决这个问题,我们使用了检测蛋白c-FLIP (抗凋亡的主要因素) 作为概念的证明。临床前环境中内源性c-FLIP水平的准确检测势在必行,因为它被认为是潜在的肿瘤生物标志物。因此,对其制造商验证并推荐用于蛋白质印迹的c-FLIP抗体的几种来源进行了严格测试。我们发现免疫识别特性存在很大差异。尽管这些抗体已在许多出版物中使用,但我们的结果表明,其中一些未能通过Western印迹检测到内源性c-FLIP蛋白。我们的结果表明,抗体验证标准是不充分的,并且系统地使用基因敲除和/或敲除来建立特异性证明是至关重要的,即使对于以前在科学文献中使用的抗体也是如此。由于抗体是临床前和临床研究的基本工具,因此确保其特异性至关重要。

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