Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells
Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells
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DOI：
10.1038/nbt.1673
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发表时间：
2010-09-01
影响因子：
46.9
通讯作者：
Georgiou, George
中科院分区：
文献类型：
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作者：
Reddy, Sai T.;Ge, Xin;Georgiou, George
抗原特异性单克隆抗体（mAb）和抗体片段的分离取决于对永生的B细胞（1,2）或重组抗体文库的高通量筛选（3-6）。我们通过使用高通量DNA测序和生物信息学分析来绕过筛选步骤，以挖掘免疫小鼠骨髓浆细胞（BMPC）的抗体可变区域（V） - 元素谱。无法永生的BMPC产生绝大多数循环抗体。我们发现，免疫后，BMPC的V-Gene曲目高度极化，最丰富的序列以与总库的1％和> 10％之间的频率表示。我们根据它们的相对频率配对了最丰富的可变重（VH）和可变光（VL）基因，并使用自动基因合成重建它们，并在细菌或哺乳动物细胞中表达重组抗体。以这种方式从六只小鼠产生的抗体，每种用三种抗原之一进行免疫的抗体都是抗原特异性的（21/27或78％）。那些由具有高血清滴度的小鼠产生的纳摩尔结合亲和力。 （c）2010 Nature America，Inc。保留所有权利。
Isolation of antigen-specific monoclonal antibodies (mAbs) and antibody fragments relies on high-throughput screening of immortalized B cells(1,2) or recombinant antibody libraries(3-6). We bypassed the screening step by using high-throughput DNA sequencing and bioinformatic analysis to mine antibody variable region (V)-gene repertoires from bone marrow plasma cells (BMPC) of immunized mice. BMPCs, which cannot be immortalized, produce the vast majority of circulating antibodies. We found that the V-gene repertoire of BMPCs becomes highly polarized after immunization, with the most abundant sequences represented at frequencies between similar to 1% and >10% of the total repertoire. We paired the most abundant variable heavy (VH) and variable light (VL) genes based on their relative frequencies, reconstructed them using automated gene synthesis, and expressed recombinant antibodies in bacteria or mammalian cells. Antibodies generated in this manner from six mice, each immunized with one of three antigens were overwhelmingly antigen specific (21/27 or 78%). Those generated from a mouse with high serum titers had nanomolar binding affinities. (C) 2010 Nature America, Inc. All rights reserved.