Design and Construction of Synthetic Phage-Displayed Fab Libraries

Diversity—the variability carried by the amino acid sequences of a synthetic antibody library—can be generated by synthetic degenerate oligonucleotides. One can experiment with different diversity designs in the variable domains of light and heavy chains (V_H and V_L ) to generate antibody libraries with different properties. The ability to precisely define the final diversity of a library facilitates the process of isolating, characterizing, and optimizing an antibody lead. Here we describe detailed protocols for the design and construction of phage-displayed synthetic antibody libraries in which diversity is generated in the complementarity determining regions (CDRs) of the V_H of a single humanized bivalent Fab scaffold. The example used in the protocol provides a general methodology for generation of libraries with engineered CDR diversity that can be applied to a template antibody sequence of choice.