食品安全质量检测学报
食品安全質量檢測學報
식품안전질량검측학보
FOOD SAFETY AND QUALITY DETECTION TECHNOLOGY
2014年
6期
1709-1717
,共9页
毒死蜱%单克隆抗体%icELISA%优化
毒死蜱%單剋隆抗體%icELISA%優化
독사비%단극륭항체%icELISA%우화
chlorprifos%monoclonal antibody%icELISA%optimization
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 IC50为73.25 ng/mL,线性范围 IC20～IC80为32.52～260 ng/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。
目的：製備毒死蜱單剋隆抗體,建立 icELISA(間接競爭酶聯免疫吸附方法)檢測方法併對其進行優化。方法以毒死蜱為基礎物質,製備半抗原CPF-H1和CPF-H2,偶聯蛋白製備人工抗原,進而通過免疫、細胞融閤、篩選得到特異性的單剋隆抗體,建立毒死蜱的icELISA檢測方法,併對其分析條件進行優化。結果質譜鑒定結果錶明毒死蜱半抗原製備成功；半抗原偶聯蛋白,紫外全波長掃描鑒定人工抗原製備成功；免疫動物、細胞融閤併製備單剋隆抗體；建立瞭icELISA檢測方法,優化得到最佳反應條件： PBST為標準品稀釋液,包被抗原最佳稀釋倍數為1：1000,抗體最佳稀釋倍數為1：1000,一抗反應最佳時間為40 min,二抗反應最佳時間為30 min,製得毒死蜱標準麯線,毒死蜱對抗體的 IC50為73.25 ng/mL,線性範圍 IC20～IC80為32.52～260 ng/mL, LOD為19.34 ng/mL。結論本研究為毒死蜱快速檢測技術奠定瞭理論基礎,對保障人們的身體健康具有重要的意義。
목적：제비독사비단극륭항체,건립 icELISA(간접경쟁매련면역흡부방법)검측방법병대기진행우화。방법이독사비위기출물질,제비반항원CPF-H1화CPF-H2,우련단백제비인공항원,진이통과면역、세포융합、사선득도특이성적단극륭항체,건립독사비적icELISA검측방법,병대기분석조건진행우화。결과질보감정결과표명독사비반항원제비성공；반항원우련단백,자외전파장소묘감정인공항원제비성공；면역동물、세포융합병제비단극륭항체；건립료icELISA검측방법,우화득도최가반응조건： PBST위표준품희석액,포피항원최가희석배수위1：1000,항체최가희석배수위1：1000,일항반응최가시간위40 min,이항반응최가시간위30 min,제득독사비표준곡선,독사비대항체적 IC50위73.25 ng/mL,선성범위 IC20～IC80위32.52～260 ng/mL, LOD위19.34 ng/mL。결론본연구위독사비쾌속검측기술전정료이론기출,대보장인문적신체건강구유중요적의의。
ABSTRACT:Objective To prepare monoclonal antibodies of chlorpyrifos, and establish and optimize icE-LISA (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.
