Selection of phage antibodies by binding affinity

We describe a process, based on display of antibodies on the surface of filamentous bacteriophage, for selecting antibodies either by their affinity for antigen or by their kinetics of dissociation (off-rate) from antigen. For affinity selection, phage are mixed with small amounts of soluble biotinylated antigen (less than 1 microgram) such that the antigen is in excess over phage but with the concentration of antigen lower than the dissociation constant (Kd) of the antibody. Those phage bound to antigen are then selected using streptavidin-coated paramagnetic beads. The process can distinguish between antibodies with closely related affinities. For off-rate selection, antibodies are preloaded with biotinylated antigen and diluted into excess unlabelled antigen for variable times prior to capture on streptavidin-coated paramagnetic beads. To mimic the affinity maturation process of the immune system, we introduced random mutations into the antibody genes in vitro using an error-prone polymerase, and used affinity selection to isolate mutants with improved affinity. Starting with a small library (40,000 clones) of mutants (average 1.7 base changes per VH gene) of the mouse antibody B1.8, and using several rounds of affinity selection, we isolated a mutant with a fourfold improved affinity to the hapten 4-hydroxy-5-iodo-3-nitrophenacetyl-(NIP)-caproic acid (mutant Kd = 9.4(+/- 0.3) nM compared with B1.8 Kd = 41.9(+/- 1.6) nm). The relative increase in affinity of the mutant is comparable to the increase seen in the anti-4-hydroxy-3-nitrophenylacetyl/NIP-caproic acid murine secondary immune response.