直接竞争化学发光酶免疫法检测氟喹诺酮类药物方法的建立  

Development of a New Detection Method of Ten Fluoroquinolones by Direct Competitive Chemiluminescent Enzyme Immunoassay

作　　者：曹敬政 安静 蒋蔚[1] 辛思培 宋斌 李思 王权[1] 孙卫东[3] CAO Jingzheng;AN Jing;JIANG Wei;XIN Sipei;SONG Bin;LI Si;WANG Quan;SUN Weidong(Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;Institute of Agricultural Quality Standards and Testing Technology,Xinjiang Academy of Agricultural Sciences,Urumqi 830000,China;College of Veterinary Medicine of Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]新疆农业科学院农业质量标准与检测技术研究所,乌鲁木齐830000 [3]南京农业大学动物医学院,南京210095

出　　处：《中国动物传染病学报》2021年第5期48-58,共11页Chinese Journal of Animal Infectious Diseases

基　　金：上海市科委项目(19391901900);自治区科技支疆项目计划(指令性)项目(2016E02028);上海市科技兴农项目(2020-02-08-00-08-F01486)。

摘　　要：建立一种能够快速检测动物源性食品中多种氟喹诺酮类药物残留的直接竞争化学发光酶免疫法。用诺氟沙星(NOR)分子修饰后制备的完全抗原免疫小鼠,获得对10种氟喹诺酮类药物具有广谱特异性的单克隆抗体,采用二因子交叉试验确定包被原与酶标抗体的最佳稀释度,通过单因素试验优化最佳包被条件、封闭液种类、竞争反应时间,从而建立反应过程的最佳条件,并对方法的灵敏度、交叉反应率、精密度进行评价。结果表明:NOR-1-OVA包被浓度0.3μg/mL,酶标抗体的稀释倍数为1∶8000时为最佳稀释浓度;4℃包被过夜,封闭液为1%BSA,竞争时间为1 h时为最优反应条件。对10种氟喹诺酮类药物的灵敏度(IC50)为1.46~11.57 ng/mL,交叉反应率为12.6%~118.9%,并且与其他非喹诺酮类药物无交叉反应率;检出限为0.07~0.27 ng/mL,且批内差异和批间差异均低于10%;加标回收率为73.3%~103.1%,变异系数为1.9%~11.8%。以上结果表明,该方法可作为动物源性食品中10种FQs残留检测的常规方法。To detect fl uoroquinolone in food-borne animal tissues,a direct competitive chemiluminescent enzyme immunoassay was developed in the present study.A complete antigen prepared by modification of norfloxacin(NOR)was used to immunize mice to prepare a monoclonal antibody having broad specifi city spectrum for 10 fl uoroquinolones.The optimal dilution of the coating antigen and the enzyme-labeled antibody was determined by a two-factor crossover experiment.The high-throughput detection of ten drugs in fl uoroquinolones was completed by optimizing the conditions of coating,the type of blocking solution,and the optimal reaction time and evaluate this method in the sensitivity,crossover rate and precision.The results show that the optimal reaction conditions included 0.3 μg/mL of coating antigen NOR-1-OVA, 1∶8000 for enzyme-labeled antibody dilution, plate coating overnight at 4℃, 1%BSA for the blocking solution and 1 hour for the competitive reaction time. The results showed 1.46-11.57 ng/mL of half-inhibition (IC_(50)) for 10 detectable fl uoroquinolones, 12.6%-118.9% of the cross-reactivity and less than 0.01% of cross-reaction with other non-quinolones. The detection limits for 10 fl uoroquinolones were 0.07-0.27 ng/mL, the average recovery rates were 73.3%-103.0% and the coeffi cient of variation were 1.9%-11.8%. Therefore, this highly sensitive method might be developed as a conventional tool to detect fl uoroquinolone residues in animal-derived foods.

关 键 词：氟喹诺酮类药物 直接竞争化学发光酶免疫反应 药物检测 

分 类 号：S852.43[农业科学—基础兽医学]