Improved affinity of a chicken single-chain antibody to avian infectious bronchitis virus by site-directed mutagenesis of complementarity-determining region H3

Affinity maturation of antibodies using random or site-directed mutagenesis provides the potential to change the properties of antibodies in vitro . We used site-directed mutagenesis to improve the binding affinity of an anti-avian infectious bronchitis virus (IBV) single-chain antibody ZL.80 with low affinity. Twelve highly variable residues in the third heavy chain complementarity-determining region (CDR H3) of ZL.80 were mutated to alanine, to determine their contributions to antigen binding. Only mutations at Cys H105, Asp H106 and Val H108 inhibited its binding to IBV in indirect enzyme-linked immunosorbent assays. These three sites were then substituted with tyrosine, arginine and serine, because these were considered to be beneficial to protein-protein interactions. The mutant Val H108 Tyr demonstrated a 12.9-fold increase in affinity compared to parental ZL.80. This study demonstrates that the affinity of single-chain variable fragments can be improved by a single amino acid substitution in the CDR H3 with no change in specificity, indicating that tyrosine could play an important role in antigen binding.