Affinity Maturation by Random Mutagenesis and Phage Display

Antibodies are essential tools for research, diagnostics and therapy. However, sometimes antibodies with a most favourable specificity profile lack sufficient affinity for a desired application. Here, a method is described to increase the affinity of recombinant scFv antibody fragments based on random mutagenesis and phage display. Random mutations are inserted by performing several rounds of error-prone PCR. After construction of a mutated antibody gene library, affinity selection is performed by panning with washing conditions optimised for off-rate dependent selection. Alternatively, panning in solution with competition can be used to enrich binders with improved binding properties.