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

Abstract

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.

Introduction

The non-ionic surfactant alkylphenol polyethoxylates (APnEOs) are used widely in pesticide formulations, detergents, and industrial products for many years due to their favorable physico-chemical properties (Ying et al., 2002). 4-Nonylphenol polyethoxylate (NPnEO) is one of the most commonly used APnEO, which is produced by addition of ethylene oxide to a mixture of branched 4-nonylphenol (NP) isomers. Released into the environment or entered into sewage treatment systems, APnEOs are biodegraded, resulting in shortening of ethoxy chain and/or carboxylation at the terminal ethoxy unit (alkylphenoxy carboxylates; APECs) that ultimately release alkylphenols (APs) (Staples et al., 1999). The hydrophobicity and fish toxicity of the biodegraded products increase as an ethoxy chain length decreases (Yoshimura, 1986). It was also reported that the metabolites of APnEOs, NP diethoxylates (NP2EO), 4-nonylphenoxyacetic acid (NP1EC), and NP show estrogenic activities (Jobling and Sumpter, 1993, Routledge and Sumpter, 1996). Because of massive use of APnEOs and ubiquitous occurrence of their metabolites in the environment, monitoring of APnEOs, APECs, and APs is of great importance for risk assessment of their contaminations.

Two anti-APnEO monoclonal antibodies (mabs) MOF3-139 and AP-14 were established to quantitatively measure APnEOs and/or APs in competitive enzyme-linked immunosorbent assay (ELISA) (Goda et al., 2000, Goda et al., 2004). Interestingly, these two mabs show clearly different specificity. AP-14 cross-reacts not only with APnEOs but also with APs. However, MOF3-139 is specific to APnEOs, does not cross-react with APs. In this study we attempted to understand the molecular basis of this specificity difference between both mabs. We first prepared single-chain Fv (scFv) recombinant antibodies corresponding to two mabs each and compared amino acid sequences of variable domains of the mabs. Based on the differences in primary structures, domain-swapped, complementarity-determining region (CDR)- or framework region (FR)-grafted, and site-directed mutant scFv antibodies were produced, and the reactivity and specificity of the scFv antibodies were examined in competitive ELISA for understanding molecular basis of the reactivity and specificity of the anti-APnEO mabs.