Rapid high-throughput cloning and stable expression of antibodies in HEK293 cells

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• 作者：Jared L. Spidel ; ^{jspidel@morphotek.com} ; Benjamin Vaessen ; Yin Yin Chan ; Luigi Grasso ; J. Bradford Kline
• 关键词：Monoclonal antibody ; High-throughput cloning ; Bicistronic ; Transfection ; Stable cell line
• 刊名：Journal of Immunological Methods
• 出版年：2016
• 出版时间：December 2016
• 年：2016
• 卷：439
• 期：Complete
• 页码：50-58
• 全文大小：899 K
• 卷排序：439

文摘

Single-cell based amplification of immunoglobulin variable regions is a rapid and powerful technique for cloning antigen-specific monoclonal antibodies (mAbs) for purposes ranging from general laboratory reagents to therapeutic drugs. From the initial screening process involving small quantities of hundreds or thousands of mAbs through in vitro characterization and subsequent in vivo experiments requiring large quantities of only a few, having a robust system for generating mAbs from cloning through stable cell line generation is essential. A protocol was developed to decrease the time, cost, and effort required by traditional cloning and expression methods by eliminating bottlenecks in these processes. Removing the clonal selection steps from the cloning process using a highly efficient ligation-independent protocol and from the stable cell line process by utilizing bicistronic plasmids to generate stable semi-clonal cell pools facilitated an increased throughput of the entire process from plasmid assembly through transient transfections and selection of stable semi-clonal cell pools. Furthermore, the time required by a single individual to clone, express, and select stable cell pools in a high-throughput format was reduced from 4 to 6 months to only 4 to 6 weeks.