Isolation of HCV Neutralizing Antibodies by Yeast Display

Yeast surface display (YSD) enables efficient screening and selection of single chain variable fragments (scFvs) of heavy (V_H) and light (V_L) chains that bind to target antigen with different affinities. Assembly of a scFv library from cDNA usually involves adding different primers and linkers (Gly₄/Ser)₃ through multiple rounds of PCR amplification and purification. We describe here a simplified scFv assembly method by creating a modified YSD vector with a built-in linker that reduces the time of assembly and decreases accumulated base exchanges due to PCR errors. In addition, we describe a bias screening strategy toward maximizing novel antibodies of interest by a combination of memory B cell selection and depletion by binding to mutant antigens that do not bind to previously identified monoclonal antibodies.