Anti-Peptide Monoclonal Antibodies Generated for Immuno-Multiple Reaction Monitoring-Mass Spectrometry Assays Have a High Probability of Supporting Western blot and ELISA
Anti-Peptide Monoclonal Antibodies Generated for Immuno-Multiple Reaction Monitoring-Mass Spectrometry Assays Have a High Probability of Supporting Western blot and ELISA
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DOI：
10.1074/mcp.o114.043133
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发表时间：
2015-02-01
影响因子：
7
通讯作者：
Paulovich, Amanda G.
中科院分区：
文献类型：
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作者：
Schoenherr, Regine M.;Saul, Richard G.;Paulovich, Amanda G.
免疫亲和富集肽偶联靶向，多反应监测质谱（免疫MRM）最近已开发用于定量分析肽和蛋白质的表达。作为该技术的一部分，针对短的线性胰蛋白酶肽产生抗体，这些肽非常适合通过质谱法进行检测。尽管其具有良好的分析性能，但广泛采用免疫-MRM的主要障碍是缺乏经验证的亲和试剂，因为商业抗体供应商不愿意投入资源来生产用于免疫-MRM的抗肽抗体，而常规技术的市场要大得多，特别是Western印迹和ELISA。这种不情愿的部分原因是担心产生短的线性胰蛋白酶肽序列的亲和试剂在检测全长蛋白质的传统测定中可能表现不佳。在这项研究中，我们测试了产生免疫MRM单克隆抗体（mAb）（靶向胰蛋白酶肽抗原）的可行性和成功率，这些抗体也与传统的基于蛋白质的免疫亲和技术兼容。我们生成了40种新的肽免疫MRM测定，并确定使用免疫MRM单克隆抗体进行Western印迹的交叉成功率为58%，ELISA为43%，这与传统免疫测定技术的交叉成功率相比是有利的。如果将常规和免疫MRM抗原设计策略相结合，这些成功率很可能会增加，我们建议这种综合方法的工作流程。此外，对40种新型免疫MRM测定进行了适用性分析验证，所有mAb和测定均已分别通过临床蛋白质组肿瘤分析联盟（CPTAC）抗体（http：assays.cancer.gov）和测定门户网站（http：//www.example.com）作为社区资源提供。antibodies.cancer.gov本研究还首次确定了使用重组B细胞克隆方法生成用于免疫MRM的mAb的成功率（92%），该方法比传统的杂交瘤方法快得多。
Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring-mass spectrometry (immuno-MRM) has recently been developed for quantitative analysis of peptide and protein expression. As part of this technology, antibodies are generated to short, linear, tryptic peptides that are well-suited for detection by mass spectrometry. Despite its favorable analytical performance, a major obstacle to widespread adoption of immuno-MRM is a lack of validated affinity reagents because commercial antibody suppliers are reluctant to commit resources to producing anti-peptide antibodies for immuno-MRM while the market is much larger for conventional technologies, especially Western blotting and ELISA. Part of this reluctance has been the concern that affinity reagents generated to short, linear, tryptic peptide sequences may not perform well in traditional assays that detect full-length proteins. In this study, we test the feasibility and success rates of generating immuno-MRM monoclonal antibodies (mAbs) (targeting tryptic peptide antigens) that are also compatible with conventional, protein-based immuno-affinity technologies. We generated 40 novel, peptide immuno-MRM assays and determined that the cross-over success rates for using immuno-MRM monoclonals for Western blotting is 58% and for ELISA is 43%, which compare favorably to cross-over success rates amongst conventional immunoassay technologies. These success rates could most likely be increased if conventional and immuno-MRM antigen design strategies were combined, and we suggest a workflow for such a comprehensive approach. Additionally, the 40 novel immuno-MRM assays underwent fit-for-purpose an- alytical validation, and all mAbs and assays have been made available as a resource to the community via the Clinical Proteomic Tumor Analysis Consortium's (CPTAC) Antibody (http://antibodies.cancer.gov) and Assay Portals (http://assays.cancer.gov), respectively. This study also represents the first determination of the success rate (92%) for generating mAbs for immuno-MRM using a recombinant B cell cloning approach, which is considerably faster than the traditional hybridoma approach.