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

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.