Selection of Antigen Binders from a Chicken Single-Chain Variable Fragment Library

Antibody production against an antigen of interest is highly efficient in chickens, and the use of chicken antibody libraries in phage display can result in high-affinity single-chain variable fragments (scFvs) for multiple applications. After library preparation from an animal immunized with the antigen of interest, the next step involves the identification of antigen binders. Here, we describe a process for the screening of a phage display chicken library using a technique called bio-panning. It consists of several rounds of binding scFv-displaying phage to antigens, followed by washing, elution, and reamplification. We also describe the steps for assessing clone pools obtained after bio-panning via an ELISA-based procedure known as “phage ELISA” to identify single clones. Last, we provide the steps for using high-throughput sequencing to analyze the pool of selected clones.