Fluorescence-activated cell sorting (FACS) in combination with yeast surface display has emerged as a vital tool for the isolation and engineering of antibodies and antibody-derived fragments from synthetic, naïve, and immune libraries. However, the generation of antibodies against certain human antigens from immunized animals, e.g., mice, can remain challenging due to the homology to the murine counterpart. Due to the phylogenetic distance from humans, avian immunization can be a powerful technique for the generation of antibodies with high specificity against human antigens. Additionally, the peculiar Ig gene diversification in chickens enables the amplification of heavy and light chain genes utilizing single primer pairs, resulting in a convenient library generation. Herein, we describe the protocol for the construction of a single chain fragment variable (scFv) library derived from chickens after immunization with epidermal growth factor receptor (EGFR) for subsequent yeast surface display as well as the screening process utilizing FACS for the isolation of high-affinity antibodies.
Keywords: Chicken antibody; Fluorescence-activated cell sorting; Immune antibody library; Library generation; Yeast surface display; scFv.