Abstract
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 (Gly(4)/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.