ABSTRACT

Antibody discovery using invitro display technologies such as phage and/or yeast display has become acornerstone in many research and development projects, including the creation of new drugs for clinical use. Traditionally, after the selection phase, random clones are isolated for binding validation and Sanger sequencing. More recently, next-generation sequencing (NGS) technology has allowed deeper insight into the antibody population after aselection campaign, enabling the identification of many more specific binders. However, this approach only provides the DNA sequences of potential binders, the properties of which need to be fully elucidated by obtaining corresponding clones and expressing them for further validation. Here we present arapid novel method to harvest potential clones identified by NGS that uses asimple PCR and yeast recombination approach. The protocol was tested in selections against three different targets and was able to recover clones at an abundance level that would be impractical to identify using traditional methods.

摘要

使用体外抗体发现诸如噬菌体和/或酵母菌展示之类的展示技术已成为许多研发项目的基础，包括开发用于临床的新药。传统上，在选择阶段之后，会分离随机克隆以进行结合验证和Sanger测序。最近，新一代测序（NGS）技术允许在选择运动后更深入地了解抗体群体，从而能够鉴定出许多更特异性的结合物。但是，该方法仅提供了潜在结合剂的DNA序列，需要通过获得相应的克隆并表达它们以进行进一步验证来充分阐明其特性。在这里，我们提出了一种新的快速方法来收集由NGS鉴定的潜在克隆，该方法使用asimple PCR和酵母重组方法。