Methods for the generation of chicken monoclonal antibody fragments by phage display

Phage display has become an important approach for the preparation of monoclonal antibodies from both immune and nonimmune sources. This approach allows for the rapid selection of monoclonal antibodies without the restraints of the conventional hybridoma approach. Although antibodies to a wide variety of antigens have been selected using phage display, some highly conserved mammalian antigens have proven to be less immunogenic in mammalian animals commonly used for immunization. In order to optimize methods for constructing chicken immunoglobulin phage display libraries in the pComb3 system, we have immunized chickens with the hapten fluorescein, and generated combinatorial antibody libraries from spleen and bone marrow RNA. Herein we present methods for the isolation of scFv, diabody and Fab fragment libraries from chickens. Chicken Fab fragment libraries are constructed using human constant regions, facilitating detection with readily available reagents as well as humanization. Analysis of the selected V-genes revealed that gene conversion events were more extensive in light-chain variable region genes as compared to heavy-chain variable region genes. In addition, we present a new variant of the pComb3 phage display vector system.