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

摘要

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?ValH108Tyr?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.