antibody maturation

Subsequent library screening will fish out the antibody mutants that have high affinity. Two library screening strategies are available. In the first "surface-panning" strategy, decreasing concentrations of antigen is surface immobilized. In the second "solution-sorting" strategy, in which a labeled antigen in solution is used, we have two approaches, selection based on the equilibrium constant (Kd) and selection based on binding kinetics. In the first approach, sub-library phage is incubated with biotinylated antigen at controlled concentrations and bound phages are captured by immobilized NeutrAvidin. Selection based on binding kinetics is also termed off-rate (Koff) selection, in which phage population is allowed to saturate the labeled antigen before a large molar excess of unlabeled antigen is added to the mix for controlled periods of time. This allows the selection of mutant antibodies that have slower off-rates. Since a reduction in Koff usually results in a higher affinity, this selection approach singles out antibody variants with improved Kd.