鸡蛋中氟苯尼考残留的AlphaLISA检测方法的建立  

Establishment of an AlphaLISA Detection Method for Florfenicol Residues in Eggs

作　　者：孙志文 王忠 于莹 赵营 张晶晶 魏紫嫣 方芳 马立才 SUN Zhiwen;WANG Zhong;YU Ying;ZHAO Ying;ZHANG Jingjing;WEI Ziyan;FANG Fang;MA Licai(Beijing Agriculture Product Quality and Safety Center,Beijing 100029,China;Agricultural and Animal Husbandry Technology Extension Center of Hailaer District in Hulunbuir City,Hulunbuir 021000,China;Beijing WDWK Biotechnology Co.,Ltd.,Beijing 100095,China)

机构地区：[1]北京市农产品质量安全中心,北京朝阳100029 [2]呼伦贝尔市海拉尔区农牧技术推广中心,内蒙古呼伦贝尔021000 [3]北京维德维康生物技术有限公司,北京海淀100095

出　　处：《中国兽医杂志》2025年第4期11-19,共9页Chinese Journal of Veterinary Medicine

基　　金：北京市良种蛋鸡产业集群鸡蛋质量安全监测项目。

摘　　要：本试验旨在建立可检测鸡蛋中氟苯尼考(FF)残留的均相光激化学发光免疫分析方法(AlphaLISA)。通过化学方法合成FF生物素半抗原,利用O-羧甲基羟胺法偶联FF抗体与受体微球,通过筛选确定鸡蛋样品稀释倍数,采用棋盘法确定FF抗体标记受体微球和FF生物素半抗原的浓度,最后基于FF抗体标记受体微球、FF生物素半抗原和链霉亲和素修饰供体微球,优化建立了检测鸡蛋中FF残留的AlphaLISA检测方法,并评价其灵敏度、特异性、准确性和重复性,及与国家标准方法GB 31658.20—2022《动物性食品中酰胺醇类药物及其代谢物残留量的测定液相色谱-串联质谱法》的一致性。质谱分析结果显示,FF生物素半抗原与目标分子的分子量一致(636 Da)。经评估,鸡蛋样品离心上清液采用25倍稀释方案。建立的AlphaLISA方法对鸡蛋中FF的检测限为0.57μg/kg,定量限为0.92μg/kg;添标回收率介于87.00%~99.50%,日内和日间变异系数均低于5%;与氟苯尼考胺的交叉反应率较高(93.27%),与12种常见兽药的交叉反应率极低(<0.1%);与国家标准方法比较,2种方法在5个添加浓度点的检测结果高度一致(R2=0.997)。结果表明,本试验建立的AlphaLISA具有高灵敏度、准确性、重复性和操作简便等优点,适用于鸡蛋中FF残留的快速检测,为鸡蛋安全监测提供了高效且可行的方案。This study aimed to develop an amplified luminescent proximity homogeneous assay linked immune sorbent assay(AlphaLISA)for detecting florfenicol(FF)residues in eggs.A FF biotin hapten was synthesized through chemical methods.The FF antibody was conjugated with acceptor beads using the O-carboxymethylhydroxylamine method.The dilution factor for egg samples was determined by screening,while the concentrations of FF antibody-labeled acceptor bead and FF biotin hapten were optimized using a checkerboard titration method.Based on the FF antibody-labeled acceptor bead,FF biotin hapten,and streptavidin-modified donor beads,the AlphaLISA method for detecting FF residues in eggs was established and evaluated for sensitivity,specificity,accuracy,repeatability,and consistency with the national standard method GB 31658.20—2022"Determination of the residues of amphenicols and their metabolites in animal-derived foods-Liquid chromatography-tandem mass spectrometry".Mass spectrometry analysis confirmed that the molecular weight of the FF biotin hapten matched that of the target molecule(636 Da).After optimization,a 25-fold dilution of egg sample supernatant was selected.The established AlphaLISA method had a limit of detection(LOD)of 0.57µg/kg and a limit of quantification(LOQ)of 0.92µg/kg.The spike recovery rates ranged from 87.00%to 99.50%,and both intra-day and inter-day coefficients of variation were below 5%.The method exhibited a high cross-reactivity with florfenicol amine(93.27%)but extremely low cross-reactivity(<0.1%)with 12 common veterinary drugs.Compared to the national standard method,the two methods showed a high degree of agreement across five spiked concentration levels(R²=0.997).These results indicate that the AlphaLISA method established in this study has high sensitivity,accuracy,repeatability,and ease of operation,making it suitable for rapid detection of FF residues in eggs.This provides an efficient and feasible solution for egg safety monitoring.

关 键 词：鸡蛋 氟苯尼考 均相光激化学发光免疫分析方法(AlphaLISA) 半抗原 

分 类 号：S859[农业科学—临床兽医学]