氧氟沙星单克隆抗体的制备及生物条形码检测技术的研究  

Preparation of ofloxacin monoclonal antibody and study on bio-bar codes technology

作　　者：栗慧 薛瑛辉[4] 冯亚宁 金艳丹[5] 周沫茏 魏东 LI Hui;XUE Ying-Hui;FENG Ya-Ning;JIN Yan-Dan;ZHOU Mo-Long;WEI Dong(Hebei Key Laboratory of Quality&Safety Analysis-Testing for Agro-Products and Food,Hebei North University,Zhangjiakou 075000,China;Zhang Jiakou Key Laboratory of Quality&Safety for Charactenistics Agro-Products,Hebei North University,Zhangjiakou 075000,China;College of Agriculture and Forestry Science and Technology of Hebei North University,Zhangjiakou 075000,China;The First Affiliated Hospital of Hebei North University,Zhangjiakou 075000,China;Chengde City Center for Disease Control and Prevention,Chengde 067000,China)

机构地区：[1]河北北方学院河北省农产品食品质量安全分析检测重点实验室,张家口075000 [2]河北北方学院张家口特色农产品质量安全重点实验室,张家口075000 [3]河北北方学院农林科技学院,张家口075000 [4]河北北方学院附属第一医院,张家口075000 [5]承德市疾病预防控制中心,承德067000

出　　处：《食品安全质量检测学报》2023年第9期169-176,共8页Journal of Food Safety and Quality

基　　金：河北省重点研发计划项目(19225504D);河北省农产品食品质量安全分析检测重点实验室绩效补助经费项目(22567613H);河北北方学院省属高校基本科研业务费项目(JYT2022010)。

摘　　要：目的制备氧氟沙星单克隆抗体,采用生物条形码技术测定氧氟沙星残留量。方法采用碳二亚胺法合成氧氟沙星完全抗原后,用免疫原免疫小鼠,将特异性强的小鼠脾细胞与骨髓瘤细胞融合。通过杂交瘤细胞的多次“筛选-亚克隆”过程,制备氧氟沙星单克隆抗体。其次,制备纳米金复合探针、磁性纳米探针,建立基于荧光定量聚合酶链式反应(polymerase chain reaction,PCR)的生物条形码检测方法,在最优条件下采用荧光定量PCR生物条形码检测方法间接测定动物源性食品中氧氟沙星残留量。结果氧氟沙星线性回归方程为Y=14.4263logX+0.09184,r^(2)=0.96。在0.1~128.0 ng/mL范围内,检出限为1.14 ng/mL,添加回收实验结果显示,氧氟沙星平均回收率和相对标准偏差分别为85.8%~102.6%、1.8%~2.6%。结论基于荧光定量PCR生物条形码免疫分析方法检测氧氟沙星的结果可靠,能够应用于实际样品的检测,为氧氟沙星的检测技术提供新的方向。Objective To prepare ofloxacin monoclonal antibody,and determine the residue of ofloxacin by biological bio-bar codes technology.Methods After the complete antigen of ofloxacin was synthesized by carbodiimide method,the splenocytes of mice with strong specificity were fused with myeloma cells.Monoclonal antibodies to ofloxacin were prepared by multiple“screening-subclonal”processes of hybridoma cells.Secondly,gold nano-composite probes and magnetic nanoprobes were prepared,and then a bio-bar codes assay based on fluorogenic quantitative polymerase chain reaction(PCR)was established,and the bio-bar codes assay based on fluorogenic quantitative PCR was used to indirectly determine the residue of ofloxacin in animal-derived foods under optimal conditions.Results The linear regression equation of ofloxacin was Y=14.4263logX+0.09184,r^(2)=0.96.When the concentration range was within the mass concentration range of 0.1‒128.0 ng/mL,the limit of detection was 1.14 ng/mL,and the adding recovery experiments results showed that the mean recoveries and relative standard deviations of ofloxacin were 85.8%‒102.6%,1.8%‒2.6%,respectively.Conclusion The results of the detection of ofloxacin based on the quantitative PCR biological barcode immunoassay method are reliable,which can be applied to the detection of actual samples,which provides a new direction for the detection technology of ofloxacin.

关 键 词：氧氟沙星 单克隆抗体 纳米金复合探针 磁性纳米探针 生物条形码 

分 类 号：TS207.3[轻工技术与工程—食品科学]