Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain
文献类型： 外文期刊
作者： Liu, Rui 1 ; Liang, Xiao 1 ; Xiang, Dandan 1 ; Guo, Yirong 1 ; Liu, Yihua 3 ; Zhu, Guonian 1 ;
作者机构： 1.Zhejiang Univ, Inst Pesticide & Environm Toxicol, Hangzhou 310058, Zhejiang, Peoples R China
2.Chinese Acad Trop Agr Sci, Environm & Plant Protect Inst, Haikou 571101, Peoples R China
3.Chinese Acad Forestry, Res Inst Subtrop Forestry, Hangzhou 311400, Peoples R China
关键词： triazophos;scFv;soluble expression;ELISA;SPR
期刊名称：INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES （ 影响因子：5.923； 五年影响因子：6.132 ）
ISSN： 1422-0067
年卷期： 2016 年 17 卷 6 期
页码：
收录情况： SCI
摘要： 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 x 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.
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