Molecular analysis of specificity of anti-nonylphenol polyethoxylate single-chain antibody fragments by grafting and designed point mutations

Alkylphenol polyethoxylates and alkylphenols are widely distributed contaminants in the environment. Two anti-alkylphenol polyethoxylate monoclonal antibodies MOF3-139 and AP-14 were established to measure these chemicals by enzyme immunoassays in previous studies. Interestingly, these two monoclonal antibodies showed different specificity; AP-14 cross-reacts with nonylphenoxyacetic acid and nonylphenol, whereas MOF3-139 does not. To understand the molecular basis of the difference in specificity, single-chain Fv (scFv) antibodies derived from the monoclonal antibodies were each produced in Escherichia coli cells and characterized in competitive enzyme-linked immunosorbent assay. The scFv antibodies exhibited comparable reactivity profiles to the derived parent monoclonal antibodies. It was found that the VH domain of AP-14 play an important role in the cross-reaction when specificity tests were performed using variable domain-swapped scFv antibodies. An experiment using complementarity-determining region (CDR)-grafted scFv antibodies revealed that CDR1 and CDR2 of AP-14 are involved in the cross-reaction to nonylphenoxyacetic acid and nonylphenol, respectively. Site-directed mutagenesis was introduced in both regions and the assay revealed that 33rd Thr and 35th His in VH domain of AP-14 were highly involved in the cross-reaction with nonylphenoxyacetic acid and that 33rd Thr, 57th Asp, and 59th Glu were involved in the cross-reaction with nonylphenol. The findings herein would contribute to the antibody engineering for specificity modification and to the generation of an alkylphenol-specific recombinant antibody by antibody engineering.