中国兽医杂志2025，Vol.61Issue(4)：11-19,9.DOI:10.20157/j.cnki.zgsyzz.2025.04.002
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
摘要
Abstract
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 0.57 μg/kg and a limit of quantification(LOQ)of 0.92 μg/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Key words
egg/florfenicol/amplified luminescent proximity homogeneous assay linked immune sorbent assay(AlphaLISA)/hapten分类
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孙志文,王忠,于莹,赵营,张晶晶,魏紫嫣,方芳,马立才..鸡蛋中氟苯尼考残留的AlphaLISA检测方法的建立[J].中国兽医杂志,2025,61(4):11-19,9.基金项目
北京市良种蛋鸡产业集群鸡蛋质量安全监测项目 （）
