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.
Nishi
Kosuke
K
Research Center for Environmental Genomics, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan. kosukenishi@gmail.com
Goda
Yasuhiro
Y
Fujimoto
Shigeru
S
Inui
Hideyuki
H
Ohkawa
Hideo
H
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular; D003429:Cross Reactions / Q000276:immunology; D005026:Ethylene Glycols / Q000737:chemistry / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D010636:Phenols / Q000737:chemistry / Q000276:immunology*; D017354:Point Mutation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology*; D016415:Sequence Alignment
D000911:Antibodies, Monoclonal; D005026:Ethylene Glycols; D007135:Immunoglobulin Variable Region; D010636:Phenols; D011994:Recombinant Proteins; C021754:terics; C041594:4-nonylphenol