水产品中恩诺沙星胶体金免疫层析毛细管检测及其前处理

摘要

目的:结合胶体金标记技术和免疫层析技术建立胶体金免疫层析毛细管检测技术,用于水产品中恩诺沙星残留的快速现场检测.方法:以间接竞争的实验原理,通过优化检测区抗原、质控区二抗、金标一抗检测浓度初步建立胶体金免疫层析毛细管检测方法,并进一步确定其检测灵敏度、特异性、重复性等及前处理的优化操作.结果:该方法检测恩诺沙星的视觉检测限为5 ng/mL,半定量检测限为1.29 ng/mL.在大菱鲆、鲅鱼、舌鳎、南美白对虾阴性样品中的平均添加回收率在78.3％～129％之间,相对标准偏差均小于10％.特异性实验表明,除对环丙沙星外,对其他相似物的特异性较好;环丙沙星的视觉检测限为10 ng/mL,半定量检测限为4.37 ng/mL,满足国标检测的限量要求.同时,针对免疫层析毛细管检测的前处理方法进行初步探究,最终确定采用常温条件下磺基水杨酸提取的前处理方法.结论:恩诺沙星胶体金免疫层析毛细管检测技术具有简单、快速、易操作、重复性好的优势,为食品药物残留检测领域提供了检测新方法.同时,选择新型试剂作为胶体金免疫层析毛细管检测技术的前处理方法,该方法可以为多种快检技术的前处理提供理论基础与技术支撑.%Objective:To establish a nano-gold capillary immunochromatographic assay (CICA) based on a combination of colloidal-gold labeling and Immunochromatography for the rapid field detection of enrofloxacin (EF) residues in aquatic products.Methods:Based on the principle of indirect competitive immunoreaction,the CICA method was developed by optimizing antigen concentration in the test area,secondary antibody in the control area and gold-labeled antibody standard concentration.Then the sensitivity,specificity and repeatability of this method and several sample pretreatments were evaluated.Results:The visual and semi-quantitative detection limits for EF were estimated to be 5 and 1.29 ng/mL,respectively.The recoveries of EF in spiked negative samples of turbot,Spanish mackerel,tonguefishes,and Pacific while shrimp (P.vannamei) were 78.3％-129％ and The intra-and inter-assay precision expressed as relative standard deviation (RSD) was less than 10％.Specificity analysis indicated that this method had strong specificity to all analogues except ciprofloxacin (CIP),for which the visual and semi-quantitative detection limit were 10 and 4.37 ng/mL,respectively and met the requirement of the national standard of China.Sulfonic acid extraction under normal temperature conditions was chosen as the optimal pretreatment method for CICA.Conclusion:CICA was a simple,rapid,easy-to-operate and reproducible method and could provide a new approach for the detection of antibiotic residues in aquatic products.Meanwhile,the pretreatment method for CICA chosen in this study can provide a theoretical basis and technical support for other rapid detection methods.