毒死蜱单克隆抗体制备及icELISA检测方法优化

目的制备毒死蜱单克隆抗体,建立icELISA(间接竞争酶联免疫吸附方法)检测方法并对其进行优化。方法以毒死蜱为基础物质,制备半抗原CPF-H1和CPF-H2,偶联蛋白制备人工抗原,进而通过免疫、细胞融合、筛选得到特异性的单克隆抗体,建立毒死蜱的icELISA检测方法,并对其分析条件进行优化。结果质谱鉴定结果表明毒死蜱半抗原制备成功;半抗原偶联蛋白,紫外全波长扫描鉴定人工抗原制备成功;免疫动物、细胞融合并制备单克隆抗体;建立了icELISA检测方法,优化得到最佳反应条件:PBST为标准品稀释液,包被抗原最佳稀释倍数为1:1000,抗体最佳稀释倍数为1:1000,一抗反应最佳时间为40 min,二抗反应最佳时间为30 min,制得毒死蜱标准曲线,毒死蜱对抗体的IC_(50)为73-25 ng/mL,线性范围IC_(20)~IC_(80)为32.52~260ng/mL,LOD为19.34 ng/mL。结论本研究为毒死蜱快速检测技术奠定了理论基础,对保障人们的身体健康具有重要的意义。

Preparation of chlorprifos monoclonal antibody and optimization of icELISA detection method

Objective To prepare monoclonal antibodies of chlorpyrifos, and establish and optimize icELISA (indirect competitive enzyme-linked immunosorbent assay) detection methods. Methods Hapten CPF-H1 and CPF-H2 were prepared based on chlorpyrifos, artificial antigen were prepared through conjugated protein, and then specific monoclonal antibodies were obtained through immunization, cellfusion and screening. icELISA of chlorpyrifosis was established, and its analysis conditions were optimized. Results Mass spec-trometry results indicated that the chlorpyrifos hapten was successfully prepared; hapten conjugated proteins, UV wavelength scanning verified the successful preparation of artificial antigen; animals were immunized, cells were fusioned and monoclonal antibodies were prepared; icELISA detection method was established, the optimal reaction conditions were optimized to be: PBST was used as the standard diluent, the optimal coating antigen dilution was 1:1000, the optimal antibody dilution was 1:1000 , the optimal reaction time of antibody was 40 min, the optimal HRP-IgG response time was 30 min, the standard curve of chlorpyrifos was prepared, the chlorpyrifos antibody IC50 was 73.25 ng/mL, the linear range of IC20~IC80 was 32.5?260 ng/mL, and the LOD was 19.34 ng/mL. Conclusion The paper laid theoretical foundation for chlorpyrifos rapid detection, which is of great significance for the protection of people's health.