title: Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain.
abstract: Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv) antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb) against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL) to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10(-10) M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples.
authors.affiliation: Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, China. jianhanjojo@163.com.
authors.forename: Rui
authors.initials: R
authors.lastname: Liu
authors.affiliation: Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, China. liangxiaozju@126.com.|Environment and Plant Protection Institute, Chinese Academy of Tropical Agriculture Sciences, Haikou 571101, China. liangxiaozju@126.com.
authors.forename: Xiao
authors.initials: X
authors.lastname: Liang
authors.affiliation: Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, China. xiangdd@zju.edu.cn.
authors.forename: Dandan
authors.initials: D
authors.lastname: Xiang
authors.affiliation: Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, China. yirongguo@zju.edu.cn.
authors.forename: Yirong
authors.initials: Y
authors.lastname: Guo
authors.affiliation: Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou 311400, China. liuyihua813@163.com.
authors.forename: Yihua
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, China. zhugn@zju.edu.cn.
authors.forename: Guonian
authors.initials: G
authors.lastname: Zhu
mesh_terms: D000915:Antibody Affinity; D000918:Antibody Specificity; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D006825:Hybridomas; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007146:Immunoglobulin lambda-Chains / Q000235:genetics* / Q000276:immunology / Q000494:pharmacology*; D063086:Organothiophosphates / Q000037:antagonists & inhibitors*; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology / Q000494:pharmacology*; D020349:Surface Plasmon Resonance; D014230:Triazoles / Q000037:antagonists & inhibitors*
chemical_list: D007143:Immunoglobulin Heavy Chains; D007146:Immunoglobulin lambda-Chains; D063086:Organothiophosphates; D057127:Single-Chain Antibodies; D014230:Triazoles; C012584:triazophos
keywords: ELISA; SPR; scFv; soluble expression; triazophos