环丙沙星酶联免疫分析方法的建立  

Development of an Enzyme Linked Immunoassay for Ciprofloxacin

作　　者：陆宁 蒲顺昌 邢爽 孙玉喜 丁德杰 屈业锋 张志祥 张慧敏 LU Ning;PU Shunchang;XING Shuang;SUN Yuxi;DING Dejie;QU Yefeng;ZHANG Zhixiang;ZHANG Huimin(Biology and Food Engineering Department,Bozhou University,Bozhou 236800,China;Bozhou Key Laboratory of Functional Food with Medicine Food Homology,Bozhou 236800,China)

机构地区：[1]亳州学院生物与食品工程系,安徽亳州236800 [2]药食同源功能食品亳州市重点实验室,安徽亳州236800

出　　处：《安徽科技学院学报》2022年第2期48-54,共7页Journal of Anhui Science and Technology University

基　　金：安徽省高校自然科学研究项目(KJ2020A0767);亳州市重点研发计划项目(bzzc2020018);亳州学院自然科学研究项目(BYZ2019C02,BY2019XZ02)。

摘　　要：目的:建立检测环丙沙星药物残留的酶联免疫分析方法。方法:采用活泼酯法制备免疫原环丙沙星-牛血清蛋白偶联物、同源包被原环丙沙星-鸡卵清蛋白偶联物、异源包被原氧氟沙星-鸡卵清蛋白偶联物。采用紫外吸收光谱法扫描鉴定人工抗原是否偶联成功。用免疫原免疫新西兰大白兔得到多克隆抗体,对其性能进行鉴定。通过间接竞争酶联免疫分析方法筛选最佳包被原,对抗体和包被原的最佳工作浓度、封闭液浓度等条件进行优化,建立检测环丙沙星的检测方法。最后对方法特异性和准确度进行评价。结果:同源包被比异源包被效果好。当包被原稀释8000倍,抗体稀释4000倍时,对环丙沙星的检测灵敏度最高,半抑制浓度IC_(50)为0.16 ng/mL,检测限IC_(10)为0.012 ng/mL。除诺氟沙星外,对其余几种环丙沙星类似物的交叉反应率均在15%以下,鱼肉、禽蛋样品的添加回收率范围为80%~105%。结论:该检测方法对环丙沙星、诺氟沙星具有较强的特异性及较高的灵敏度,可用于水产品、禽蛋中环丙沙星、诺氟沙星的快速检测。Objective:To develop an enzyme-linked immunosorbent assay for the detection for the determination of Ciprofloxacin residues.Methods:Immunogenic ciprofloxacin bovine serum protein conjugate,homologous coating antigen ciprofloxacin chicken ovalbumin conjugate and heterologous coating antigen ofloxacin chicken ovalbumin conjugate were prepared by active ester method.Ultraviolet absorption spectroscopy was used to scan and identify whether the artificial antigen was successfully coupled.The polyclonal antibody was obtained by immunizing New Zealand white rabbits with immunogen,and its performance was identified.Indirect competitive enzyme-linked immunosorbent assay was used to screen the best coating agent,optimize the optimal working concentration,blocking solution concentration of antibody and coating agent,and establish a detection method for ciprofloxacin.Finally,the specificity and accuracy of the method were evaluated.Results:The effect of homologous coating was better than that of heterologous coating.When the coating was diluted 8000 times and the antibody was diluted 4000 times,the detection sensitivity of ciprofloxacin was the highest.The semi-inhibitory concentration IC_(50) was 0.16 ng/mL and the detection limit IC_(10) was 0.012 ng/mL.Except norfloxacin,the cross-reaction rates of other ciprofloxacin analogues were less than 10%.The recoveries of fish and poultry eggs ranged from 80%to 105%.Conclusion:The method has strong specificity and sensitivity for ciprofloxacin and norfloxacin.It can be used for the rapid determination of ciprofloxacin and norflo xacin in aquatic products and poultry eggs.

关 键 词：环丙沙星 同源包被 间接竞争ELISA 

分 类 号：TS207.3[轻工技术与工程—食品科学]