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

Abstract

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 ^CysH105, ^AspH106 and ^ValH108 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 ^ValH108^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.

Key words: Single-chain variable fragments (scFvs), affinity maturation, avian infectious bronchitis virus (IBV), third heavy chain complementarity-determining region (CDR H3).