抗体片段的自动化高通量纯化，以促进基于功能和动力学的分析评估。

Automated high-throughput purification of antibody fragments to facilitate evaluation in functional and kinetic based assays.

作者信息

Su Bin, Hrin Renee, Harvey Barrett R, Wang Ying-Jie, Ernst Robin E, Hampton Richard A, Miller Michael D, Strohl William R, An Zhiqiang, Montgomery Donna L

机构信息

Department of Vaccines and Biologics Research, Merck Research Laboratories, 770 Sumneytown Pike, P.O. Box 4, West Point, PA 19486, USA.

出版信息

J Immunol Methods. 2007 Apr 30;322(1-2):94-103. doi: 10.1016/j.jim.2007.02.006. Epub 2007 Mar 7.

DOI:10.1016/j.jim.2007.02.006

PMID:17362979

Abstract

Screening antibodies from phage displayed in vitro libraries and from affinity maturation of lead antibodies requires testing of antibody fragments (scFvs and Fabs) for function and binding affinities. Crude scFv or Fab periplasmic preparations from Escherichia coli are often not pure and/or concentrated enough for use in functional and affinity assays. We have developed an automated high-throughput approach for small and large-scale expression and purification of His-tagged scFvs and Fabs using the Qiagen BioRobot 3000 LS with optimized application software. This automated procedure enabled us to rapidly evaluate antibody fragments in functional and surface plasmon resonance (SPR) assays. We have used these procedures to make thousands of purified scFv/Fabs for several antibody maturation campaigns and significantly decreased the time needed to select the best candidates. The assay results from these purified samples were used to prioritize candidates before converting them to IgG. This protocol can process up to 300 small-scale and up to 72 large-scale scFvs or Fabs per week per full-time employee (FTE).

摘要

从体外展示的噬菌体文库中筛选抗体以及对先导抗体进行亲和力成熟，都需要检测抗体片段（单链抗体片段和Fab片段）的功能和结合亲和力。来自大肠杆菌的粗制单链抗体片段或Fab片段周质体制剂通常纯度不够且/或浓缩程度不足，无法用于功能和亲和力测定。我们开发了一种自动化的高通量方法，使用配备优化应用软件的Qiagen BioRobot 3000 LS，用于小规模和大规模表达及纯化带有His标签的单链抗体片段和Fab片段。这种自动化程序使我们能够在功能和表面等离子体共振（SPR）测定中快速评估抗体片段。我们已使用这些程序为多个抗体成熟项目制备了数千个纯化的单链抗体片段/Fab片段，并显著减少了选择最佳候选抗体所需的时间。这些纯化样品的测定结果用于在将候选抗体转化为IgG之前对其进行优先级排序。该方案每名全职员工（FTE）每周最多可处理300个小规模和72个大规模的单链抗体片段或Fab片段。