Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications.
Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications.
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抗体验证程序的扩展可以量化主要研究应用中的抗体性能。
DOI：
10.1101/2023.06.01.543292
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Ayoubi R
中科院分区：
文献类型：
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作者：
Ayoubi R
抗体是检测和鉴定人类蛋白质的关键试剂。生物医学研究界渴望为每种人类蛋白质和每种常见应用至少有一种有效的和选择性的可再生抗体。为了量化商用试剂抗体实现这一目标的可能性，我们应用标准化的表征方法来评估针对65个神经科学相关蛋白的614个商用抗体在蛋白质印迹、免疫沉淀和免疫荧光应用中的性能，并以同基因敲除细胞为对照。通过对抗体进行并列比较，我们证明：i)对于所测试的每一种应用，该蛋白质组的61%至89%被至少一种高效抗体覆盖，其中54%至77%被至少一种高效可再生抗体覆盖，这表明商用抗体对人类蛋白质的覆盖是显著的；ii)重组抗体的平均表现优于单克隆或多克隆抗体；iii)已发表的文章中使用了数百种表现不佳的抗体。令人鼓舞的是，在没有达到预期效果的商业抗体中，18%的抗体被制造商从市场上撤下，37%的抗体改变了推荐的用法。这项工作表明，实现用选择性可再生抗体覆盖人类蛋白质组的第一步是挖掘现有的商业抗体库，然后利用这些知识来集中新的可再生抗体的生成工作。
Antibodies are critical reagents to detect and characterize human proteins. The biomedical research community aspires to have at least one potent and selective renewable antibody for each human protein, and for each of the common applications. To quantify the potential to achieve this goal with commercial reagent antibodies, we applied a standardized characterization approach to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins in Western blot, immunoprecipitation, and immunofluorescence applications, using isogenic knockout cells as controls. By carrying out side-by-side comparisons of the antibodies, we demonstrate that: i) for each application tested, 61% to 89% of this protein set was covered by at least one high-performing antibody with 54% to 77% covered by at least one high-performing renewable antibody, suggesting that coverage of human proteins by commercial antibodies is significant; ii) on average recombinant antibodies performed better than monoclonal or polyclonal antibodies; iii) hundreds of underperforming antibodies have been used in published articles. Encouragingly, of the commercial antibodies that did not perform as expected, 18% were removed from the market by manufacturers and 37% had alterations made to their recommended usage. This work suggests that the first step toward achieving coverage of the human proteome with selective renewable antibodies is to mine the existing commercial antibody repertoire, and then use this knowledge to focus new renewable antibody generation efforts.