Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning
Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning
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DOI：
10.1016/j.jim.2007.09.017
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发表时间：
2008-01-01
影响因子：
2.2
通讯作者：
Wardemann, Hedda
中科院分区：
文献类型：
--
作者：
Tiller, Thomas;Meffre, Eric;Wardemann, Hedda
我们已经开发了一种有效的策略，将免疫球蛋白（Ig）基因库分析和单细胞水平的Ig反应性分析相结合。基于表面标记的表达，通过荧光激活细胞分选分离人B细胞发育不同阶段的单个细胞。通过巢式RT-PCR扩增每个细胞Ig重链和相应的Ig轻链基因转录本，并将其克隆到真核表达载体中，在体外产生具有相同特异性的单克隆人抗体。所有的反应都在96孔板中进行，可以克隆大量的Ig基因。重组抗体与多种自身和非自身抗原进行了反应性测试，反应性谱可以直接与完整的Ig重链和Ig轻链基因序列信息联系起来，这是克隆策略的一部分。总之，我们的克隆和表达人单克隆抗体的方法是无偏的，高效的，只需要小细胞数量，重组抗体可以直接得出在不同的库中特定的人B细胞频率的结论。(C) 2007 Elsevier B.V.版权所有
We have developed an efficient strategy that combines immunoglobulin (Ig) gene repertoire analysis and Ig reactivity profiling at the single cell level. Based on surface marker expression individual cells at different stages of human B cell development are isolated by fluorescence-activated cell sorting. For each cell Ig heavy and corresponding Ig light chain gene transcripts are amplified by nested RT-PCR and cloned into eukaryotic expression vectors to produce monoclonal human antibodies of the same specificity in vitro. All reactions are performed in 96-well plates and allow cloning of large numbers of Ig genes. The recombinant antibodies are tested for reactivity with diverse self- and non-self antigens and the reactivity profile can be directly linked to the complete Ig heavy and Ig light chain gene sequence information that is obtained as part of the cloning strategy. In summary, our method to clone and express human monoclonal antibodies is unbiased, highly efficient, requires only small cell numbers and the recombinant antibodies allow direct conclusions on the frequency of specific human B cells in a diverse repertoire. (C) 2007 Elsevier B.V. All rights reserved.