High-throughput affinity ranking of antibodies using surface plasmon resonance microarrays

A method was developed to rapidly identify high-aYnity human antibodies from phage display library selection outputs. It combines high-throughput Fab fragment expression and puriWcation with surface plasmon resonance (SPR) microarrays to determine kinetic constants (kon and koV) for 96 diVerent 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 puriWed using protein A PhyTip columns. Kinetic constants were obtained for 191 unique anti-hK1 Fabs using the Flexchip SPR microarray device. The highest aYnity 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 aYnities of hundreds of antibodies signiWcantly accelerates the discovery of high-aYnity antibody leads.