Liu Rui, Liang Xiao, Xiang Dandan, Guo Yirong, Liu Yihua, Zhu Guonian
Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, China.
Environment and Plant Protection Institute, Chinese Academy of Tropical Agriculture Sciences, Haikou 571101, China.
Int J Mol Sci. 2016 Jun 7;17(6):823. doi: 10.3390/ijms17060823.
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.
三唑磷是一种广泛使用的有机磷杀虫剂，对生物体具有潜在的不利影响。在本研究中，开发了一种具有特定λ轻链的高亲和力单链可变片段（scFv）抗体用于残留监测。首先，从分泌抗三唑磷单克隆抗体（mAb）的杂交瘤细胞系8C10中正确扩增出特定的可变区。然后通过重叠延伸聚合酶链反应将这些区域组装成scFv。随后，重组抗三唑磷scFv-8C10在大肠杆菌菌株HB2151中成功以可溶性形式表达，通过固定化金属离子亲和色谱法纯化，并通过蛋白质印迹和肽质量指纹分析进行验证。之后，基于纯化的抗三唑磷scFv-8C10抗体建立了间接竞争酶联免疫吸附测定法。该测定法表现出与基于亲本mAb的测定法相似的特性，对三唑磷具有高灵敏度（IC50为1.73 ng/mL），对其他有机磷农药无交叉反应；在检测加标水样中的三唑磷残留时可靠。此外，使用表面等离子体共振生物传感器进行的动力学测量表明，纯化的scFv-8C10抗体具有1.8×10（-10）M的高亲和力，并表现出良好的结合稳定性。结果表明，重组高亲和力scFv-8C10抗体是一种有效的检测材料，有望用于监测环境样品中的三唑磷残留。
