牛奶中喹诺酮类药物残留的AlphaLISA检测方法的研究  

AlphaLISA Detection Method for Quinolone Residues in Milk

作　　者：马立才 邢维维 于莹 贾良曦 秦誉 金兴 丁亚芳 MA Licai;XING Weiwei;YU Ying;JIA Liangxi;QIN Yu;JIN Xing;DING Yafang(Beijing WDWK Biotechnology Co.,Ltd.,Beijing 100095,China)

机构地区：[1]北京维德维康生物技术有限公司,北京100095

出　　处：《食品工业科技》2024年第14期264-270,共7页Science and Technology of Food Industry

基　　金：石家庄市科技计划:生牛乳中危害因子高通量检测技术的研发与应用(221170243A)。

摘　　要：本文旨在建立一种可检测牛奶中喹诺酮类药物(Quinolones,QNS)残留的均相光激化学发光免疫分析方法(Amplifiedluminescent proximity homogeneous assay linkedimmune sorbent assay,AlphaLISA)。采用EDC/NHS活泼酯法在诺氟沙星分子连接6-氨基己酸作为间隔臂,制备生物素修饰诺氟沙星半抗原(Biotin-6AS-NOR),并在受体微球上偶联QNS单克隆抗体,Biotin-6AS-NOR可与牛奶样品中的QNS竞争结合喹诺酮抗体;通过优化受体微球的QNS抗体偶联量,Biotin-6AS-NOR及供/受体微球的用量等参数,以及牛奶样品的稀释方案,建立了牛奶中QNS残留的AlphaLISA检测方法。采用1:5牛奶样品稀释方案,所建立牛奶中QNS残留的AlphaLISA检测方法的检出限和定量限分别为0.19 ng/mL和0.46 ng/mL,回收率在85.97%~110.11%之间,日内和日间变异系数均小于10%,与诺氟沙星、恩诺沙星、环丙沙星、氧氟沙星、培氟沙星、达氟沙星和洛美沙星的交叉反应率均高于70%。本研究所建立AlphaLISA检测方法具有灵敏度和准确度高、重复性好等优点,且在整个检测过程中不需要繁琐洗涤操作,具有较高的应用和推广价值。This article aimed to establish an amplified luminescent proximity homogeneous assay linked immune sorbent assay(AlphaLISA)for detecting quinolones(QNS)residues in milk.The EDC/NHS active ester method was used to connect 6-aminocaproic acid as the spacer arm on the norfloxacin molecule to prepare biotin-modified norfloxacin hapten(Biotin-6AS-NOR),and QNS monoclonal antibody was coupled on the receptor microsphere.Biotin-6AS-NOR could compete with QNS in the sample to bind quinolone antibodies.A competitive indirect AlphaLISA for QNS residues in milk was established by optimizing the parameters including QNS antibody coupling amount on receptor microspheres,Biotin-6AS-NOR,the dosage of donor/receptor microspheres,and dilution schemes for milk samples.By using the 1:5 milk sample dilution scheme,the limit of detection and the limit of quantification of the established AlphaLISA for QNS in milk were 0.19 ng/mL and 0.46 ng/mL,respectively.The recovery values ranged from 85.97%and 110.11%,and the intraday and interday variation were both less than 10%.The cross-reactivity values for norfloxacin,enrofloxacin,ciprofloxacin,ofloxacin,pefloxacin,ofloxacin,and lomefloxacin were all higher than 70%.In conclusion,the established AlphaLISA showed high sensitivity,accuracy and good repeatability,without the need of tedious wash steps throughout the detection process;therefore,it is easy to achieve automated detection of chemical hazards residue in milk and has high application and promotion value.

关 键 词：喹诺酮类 半抗原 生物素 AlphaLISA 

分 类 号：TS252.7[轻工技术与工程—农产品加工及贮藏工程]