Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation
Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation
复制标题
DOI：
10.1038/nmeth.3472
复制
发表时间：
2015-08-01
期刊：
影响因子：
48
通讯作者：
Edwards, Aled M.
中科院分区：
文献类型：
--
作者：
Marcon, Edyta;Jain, Harshika;Edwards, Aled M.
抗体被用于多种细胞生物学应用，但没有标准化的方法来评估抗体质量--缺乏这种方法会危及数据的完整性和重复性。我们描述了一种基于质谱学的免疫沉淀抗体质量评分的标准操作程序。我们通过比较来自靶抗原的归一化光谱丰度因子与所有其他蛋白质的归一化光谱丰度因子，对针对152个染色质相关的人类蛋白的1,124个新重组抗体的免疫沉淀物中的所有蛋白质的丰度进行了量化。我们在五个独立实验室的盲法研究中验证了标准操作程序的表现。以目标抗原或其已知蛋白质复合体中的一员为最丰富蛋白质的抗体被归类为“IP黄金标准”。这种方法产生的定量输出可以存储在公共数据库中并存档，它代表着朝着抗体质量社区基准平台迈出的一步。
Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.