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.