Full Antigen Kinetics of Fabs Directly from Crude Periplasmic Extracts on an Anti-CH1 Capture Surface

Abstract

Characterizing detailed binding kinetics of antibody fragments directly from bacterial periplasmic extracts (PPEs) has been historically challenging because purification of all primary hits is time and resource intensive. Here we have described a highly scalable, yet data rich approach to characterizing the binding kinetics of Fab candidates directly from crude PPE sources. The throughput of the LSA^XT can measure full kinetics (k_a, k_d, and K_D) for thousands of Fabs in a single week, while eliminating the need for purification. The assay demonstrated here using 96 unique Fabs required fewer than 9 ug of antigen and was completed in 10 hrs. Importantly, this set of 96 clones is only one-twelfth of the 1152 clone capacity that can be analyzed in a single experiment. The ability to quantitate complete kinetic profiles for responses with R_max values as low as 5 RU emphasizes that data can be obtained even for poorly expressing clones and is a key benefit of the enhanced sensitivity of the LSA^XT.

Posted by Noah T. Ditto