In vivo antigen-driven plasmablast enrichment in combination with antigen-specific cell sorting to facilitate the isolation of rare monoclonal antibodies from human B cells
In vivo antigen-driven plasmablast enrichment in combination with antigen-specific cell sorting to facilitate the isolation of rare monoclonal antibodies from human B cells
复制标题
DOI：
10.1038/nprot.2014.104
复制
发表时间：
2014-07-01
期刊：
影响因子：
14.8
通讯作者：
Swem, Lee R.
中科院分区：
文献类型：
--
作者：
Lin, Zhonghua;Chiang, Nancy Y.;Swem, Lee R.
The ability to rapidly generate large panels of antigen-specific human antibodies in a rodent would enable the efficient discovery of novel therapeutically useful antibodies. We have developed a system wherein human antigen-specific antibody-secreting plasmablasts can be enriched in vivo, in a severe combined immunodeficient (SCID)/beige mouse host. The antigen-specific plasmablasts can then be sorted by flow cytometry, enabling single-cell cloning and expression of fully human immunoglobulin-G. By using this technique, we have generated four broadly reactive anti-influenza A antibodies. Therefore, the method described here is useful for the identification of rare functional antibodies. This protocol takes similar to 1 month to complete, from the time of human vaccination to the cloning of heavy-and light-chain genes. For additional small-scale transient expression, purification and binding analysis, the protocol would take an additional month.
