| Literature DB >> 16510109 |
Dina Wassaf1, Guannan Kuang, Kris Kopacz, Qi-Long Wu, Que Nguyen, Mark Toews, Janja Cosic, Judith Jacques, Steve Wiltshire, Jeremy Lambert, Csaba C Pazmany, Shannon Hogan, Robert C Ladner, Andrew E Nixon, Daniel J Sexton.
Abstract
A method was developed to rapidly identify high-affinity human antibodies from phage display library selection outputs. It combines high-throughput Fab fragment expression and purification with surface plasmon resonance (SPR) microarrays to determine kinetic constants (kon and koff) for 96 different Fab fragments in a single experiment. Fabs against human tissue kallikrein 1 (hK1, KLK1 gene product) were discovered by phage display, expressed in Escherichia coli in batches of 96, and purified using protein A PhyTip columns. Kinetic constants were obtained for 191 unique anti-hK1 Fabs using the Flexchip SPR microarray device. The highest affinity Fabs discovered had dissociation constants of less than 1 nM. The described SPR method was also used to categorize Fabs according to their ability to recognize an apparent active site epitope. The ability to rapidly determine the affinities of hundreds of antibodies significantly accelerates the discovery of high-affinity antibody leads.Entities:
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Year: 2006 PMID： 16510109 DOI： 10.1016/j.ab.2006.01.043
Source DB: PubMed Journal: Anal Biochem ISSN： 0003-2697 Impact factor: 3.365