Molecular Immunology 46 (2009) 3125–3130 Contents lists available at ScienceDirect Molecular Immunology journal homepage: www.elsevier.com/locate/molimm Molecular analysis of specificity of anti-nonylphenol polyethoxylate single-chain antibody fragments by grafting and designed point mutations Kosuke Nishi a,∗ , Yasuhiro Goda b , Shigeru Fujimoto b , Hideyuki Inui a , Hideo Ohkawa a,1 a Research Center for Environmental Genomics, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan b Life-Environment Division, Takeda Chemical Industries, Ltd., 2-17-85, Juso-Honmachi, Yodogawa-ku, Osaka 532-8686, Japan article info Article history: Received 12 May 2009 Accepted 30 May 2009 Available online 9 July 2009 Keywords: scFv ELISA Specificity modification Alkylphenols Alkylphenol polyethoxylates 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 mon- oclonal 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 speci- ficity, 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. © 2009 Elsevier Ltd. All rights reserved. 1. 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 envi- ronment or entered into sewage treatment systems, APnEOs are biodegraded, resulting in shortening of ethoxy chain and/or car- boxylation 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 ∗ Corresponding author. Present address: Industry and Academic Coopera- tion Laboratory for Drug Development, Mokpo National University, 61 Dorim-ri, Cheonggye-myeon, Muan, Jeonnam 534-729, Republic of Korea. Tel.: +82 61 450 6293; fax: +82 61 281 1732. E-mail address: kosukenishi@gmail.com (K. Nishi). 1 Present address: Research Center for Green Science, Fukuyama University, 1 Gakuen-cho, Fukuyama, Hiroshima 729-0292, Japan. 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, 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 com- pared 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 pro- 0161-5890/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2009.05.182