A proteomics approach for the identification and cloning of monoclonal antibodies from serum
A proteomics approach for the identification and cloning of monoclonal antibodies from serum
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DOI：
10.1038/nbt.2167
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发表时间：
2012-05-01
影响因子：
46.9
通讯作者：
Polakiewicz, Roberto D.
中科院分区：
文献类型：
--
作者：
Cheung, Wan Cheung;Beausoleil, Sean A.;Polakiewicz, Roberto D.
我们描述了一种蛋白质组学方法，该方法直接从免疫动物血清中的循环多克隆抗体中识别抗原特异性抗体序列。该方法涉及具有高比活性的抗体的亲和纯化，然后通过纳米流液相色谱耦合串联质谱分析消化的抗体级分。抗体可变区的高置信度肽谱匹配通过搜索由免疫动物的B细胞免疫球蛋白库的下一代DNA测序创建的参考数据库来获得。最后，将重链和轻链序列配对并表达为重组单克隆抗体。利用该技术，我们从免疫的兔和小鼠血清中分离了针对五种抗原的单克隆抗体。单克隆抗体的抗原特异性活性重现或超过原始亲和纯化的多克隆抗体。这项技术可能有助于发现和开发疫苗和抗体疗法，并帮助我们更深入地了解体液反应。
We describe a proteomics approach that identifies antigen-specific antibody sequences directly from circulating polyclonal antibodies in the serum of an immunized animal. The approach involves affinity purification of antibodies with high specific activity and then analyzing digested antibody fractions by nano-flow liquid chromatography coupled to tandem mass spectrometry. High-confidence peptide spectral matches of antibody variable regions are obtained by searching a reference database created by next-generation DNA sequencing of the B-cell immunoglobulin repertoire of the immunized animal. Finally, heavy and light chain sequences are paired and expressed as recombinant monoclonal antibodies. Using this technology, we isolated monoclonal antibodies for five antigens from the sera of immunized rabbits and mice. The antigen-specific activities of the monoclonal antibodies recapitulate or surpass those of the original affinity-purified polyclonal antibodies. This technology may aid the discovery and development of vaccines and antibody therapeutics, and help us gain a deeper understanding of the humoral response.