悬浮芯片法与常规酶联免疫吸附法检测3种兽药残留的比较

悬浮芯片法与常规酶联免疫吸附法检测3种

兽药残留的比较

作者：刘楠苏璞高志贤朱茂祥杨陟华潘秀颉晁福寰

【摘要】建立氯霉素，克伦特罗和雌二醇 3种兽药残留同时检测的悬浮芯片法。通过将3种兽药的BSA蛋白结合物偶联于悬浮芯片的固相载体——聚苯乙烯荧光微球上作为检测探针，采用间接竞争法，在液相反应体系中，3种小分子兽药抗原和微球上的兽药结合物共同竞争液相中各自特异性的生物素化单抗，再加入藻红蛋白标记的链霉亲和素，反应后检测获得荧光信号，绘制出3种兽药残留检测的标准曲线。同时进行3种兽药的常规酶联免疫吸附法标准曲线的测定。在检测技术、检出限、检测区间、特异性、盲样测定和多元分析等方面对两种方法进行比较。除了特异性外的其它指标的比较中，悬浮芯片法均具有明显优势。两种方法的特异性检测具有良好的一致性。高通量悬浮芯片技术，具有操作简单、灵敏快速和成本低廉等优点，为多种兽药残留的快速检测提供了新方法。

【关键词】悬浮芯片，酶联免疫吸附法，残留，微球，中位荧光强度值

Abstract A novel suspension array technology was established for the detection of three kinds of veterinary drug residues: chloramphenicol, clenbuterol and 17βestradiol. The three conjugates in which veterinary drugs coupled with BSA were immobilized on the solid carrier of the suspension

microarray polystyrene fluorescent microspheres/beads as detective probes. Indirect competitive technology was employed. Competitive reactions between the veterinary drugs in the aqueous phase and the veterinary drugs BSA conjugates on the beads for coupling with their complimentary specific biotinylated monoclonal antibodies were carried out. And then, straptavidin phycoerythrin was added for coupling and the fluorescent signals were captured. Afterwards the detective standard curves were plotted. The regular ELISA standard curves of the three veterinary drugs were also plotted. Comparison between suspension array and regular enzyme linked immunosorbent assay(ELISA) was in the respects of the detective technology, the detection limits, the detective ranges, the samples detection and the multi analysis. Suspension array technology is distinct advantageous except for specificity. There was well consistent performance between the two methods. The high throughput suspension array provides a novel method for multi analysis of veterinary drugs with simple operation, sensitive, rapid and low costing.

Keywords Suspension microarray, Enzyme linked immunosorbent assay, residue, microspheres beads, median fluorescent intensity

1 引言

近年来，食品安全已成为社会共同关注的公共卫生问题，而兽药残留成为食品安全监管的重要问题。兽药残留如克伦特罗(clenbuterol, CL)、氯霉素(chloramphenicol, CAP)和雌二醇 (17

βestradiol, E2)的滥用和食用可导致多种健康损害[1～6]。加强兽药残留检测分析是监管和控制兽药残留的重要手段。因此，简单、快速和灵敏的微痕量检测技术的研究和创新尤为重要。目前，酶联免疫吸附测定法(enzyme linked immunosorbent assay, ELISA)[7,8]是兽药残留的主要检测方法之一具有简单、快速、灵敏度高和特异性强等诸多优点[9]。悬浮芯片技术是一种多功能的液相芯片分析平台，既具有固态片膜芯片的高通量特性，又具有操作简便、重复性好、灵敏度高等优点。目前，对该技术的研究多集中于大分子蛋白质和核酸的检测[10～12]，并未见应用悬浮芯片同时检测多种兽药CAP、CL和E2的报道。本研究采用悬浮芯片法和常规ELISA法对3种兽药进行同时检测，并在灵敏度、特异性、检出限等方面进行比较。

2 实验部分

2.1 仪器和试剂

Bio Plex悬浮系统，Model 680型酶标仪(美国Bio Rad公司)；MALDI TOF MS REFLEXⅢ型质谱仪(德国Bruker公司)，DU530型紫外可见分光光度计 (美国Beckman公司);6930型冷冻离心机(日本Kubota公司)；MS3型旋涡混匀器(德国IKA公司)，YM10过滤柱(孔径1.2 μm，美国Millipore公司)，Costar96孔酶标板 (美国