A method to characterize antibody selectivity immunoprecipitation, part 2

Antibodies are used in multiple cell biology applications but there are methods to assess antibody quality, riskingdata integrity and repr developed a standard operating procedure using HEK293 cells to score and antibodies for suitability in immunoprecipitation. This method, validated in in fourindependent laboratories, was developed using 1124 novel recomb for152 chromatin-related human proteins. We used mass spectrometry abundanceof all the proteinsin eachimmunoprecipitateand compared spectral abundance factors from the target antigen with those of all Antibodies for which the targetantigen or a member of its known protein most abundant protein were classified as “IP gold standard”. This m quantitative outputs that can be stored and archived in public databases, step towards a platform for community benchmarking of antibody quality.

抗体可应用于多种细胞生物学实验，但当前的抗体质量评估方法存在损害数据完整性与实验重现性的风险。本研究开发了一套基于HEK293细胞（HEK293 cells）的标准操作流程，用于筛选适配免疫沉淀（immunoprecipitation）实验的抗体。该方法已在4家独立实验室中完成验证，其构建使用了针对152种染色质相关人类蛋白的1124种全新重组抗体。我们对每一次免疫沉淀产物中所有蛋白的质谱（mass spectrometry）丰度进行了检测，并将靶抗原的光谱丰度因子（spectral abundance factors）与其余所有蛋白的该因子进行了比对。若某抗体对应的靶抗原，或其已知蛋白质复合物成员，为免疫沉淀产物中丰度最高的蛋白，则将该抗体归类为“IP金标准（IP gold standard）”抗体。本方法可生成可存储并归档至公共数据库的定量输出结果，为构建抗体质量社区基准测试平台迈出了关键一步。