H9亚型AIV型特异性电化学发光免疫检测方法的建立  

Electrochemical Luminescence Immunoassay for the Detection of H9 Subtype Avian Influenza Virus

作　　者：亓文宝[1] 李芳[1] 李华楠[1] 黄丽红[1] 和君[1] 穆光慧[1] 罗开健[1] 廖明[1] 

机构地区：[1]华南农业大学兽医学院/人兽共患病防控制剂国家地方联合工程实验室,广州510642

出　　处：《中国农业科学》2015年第15期3064-3070,共7页Scientia Agricultura Sinica

基　　金：国家肉鸡产业技术体系项目(nycytx-42-G3-03);广州市珠江科技新星专项(2014J2200072)

摘　　要：【目的】H9亚型禽流感是重要的人兽共患性传染病,该亚型病毒为H7N9亚型和H10N8亚型流感病毒提供了6个内部基因(PB2、PB1、PA、NP、M、NS),并且一直处于动态重组过程中。因此,建立针对H9亚型禽流感的型特异性电化学发光免疫的高通量快速检测方法,加强H9亚型流感的监测,具有重要意义。【方法】用钌联吡啶标记H9亚型AIV的单克隆抗体,用MPI-E型电致化学发光分析系统评价钌标单抗标记效率;用生物素标记H9亚型AIV的多克隆抗体,HABA法检测抗体生物素化的效率;待测抗原与钌标单抗作用1 h后,将此抗原-抗体复合物与通过生物素-链霉亲和素系统固定在磁微球表面的多克隆抗体反应,最后加入反应底物三丙胺后即可在电化学分析系统进行发光检测。优化生物素化多抗和钌标单抗最佳工作浓度,确定临界值和反应谱,对所建立的方法进行敏感性、特异性和重复性试验。攻毒后3 d和5 d,采集攻毒组和空白对照组鸡只的88份咽拭子和肛拭子,分别用电化学发光免疫检测方法和鸡胚病毒分离法进行检测和比较。【结果】钌标抗H9亚型AIV单克隆抗体的标记效率为每个单抗Ig G分子上结合21个Ru2+,且间接免疫荧光方法证明其仍具有生物活性;生物素化兔抗H9N2亚型AIV多克隆抗体的标记效率为每个Ig G分子上结合了6个生物素分子,且Western blotting试验证明其仍保持生物活性;该方法的检测临界值为28.3,可疑区间为23.4—33.2;阴性和阳性变异系数均小于10%;检测限为5×104EID50,能够特异性地检测H9亚型AIV,不与其他亚型流感病毒(H1、H3、H4、H5和H6亚型)和其他类型的禽源病毒(NDV、IBV和IBDV)反应。3 h内即可完成检测,与鸡胚病毒分离法的符合率为86.4%。【结论】所建立的H9亚型AIV型特异性电化学发光免疫检测方法可以用于临床样品检测,对H9亚型禽流感的监测和防控具有重要意义。【Objective】 H9 subtype avian influenza is an important zoonosis. Their six internal genes（PB2, PB1, PA, NP, M, NS） of H7N9 and H10N8 subtypes influenza viruses were derived from endemic H9N2 influenza viruses circulating in poultry. The objective of the study is to establish a special detecting method for H9 subtype avian influenza virus by Electrochemical Luminescence Immunoassay（ECLIA）. This ECLIA is significant for influenza surveillance.【Method】The monoclonal antibody and rabbit polyclonal antibody anti-H9 subtype AIV were firstly labeled with [Ru（bpy）3]2＋ and biotin, respectively. And the labeled efficient was evaluated by MPI-E system and HABA. The second step is the reaction between samples and monoclonal antibody symbolized with [Ru（bpy）3]2＋. Then combine the antigen-antibody complex with rabbit polyantibodies labled by biotin-streptavidin linkage system. The chemiluminescence detection can be conducted within the electrochemical analysis system after addition oftripropylamine as substrate. The best working concentrations of the labeled monoclonal antibody and rabbit polyclonal antibody anti-H9 subtype AIV were optimized. The sensitivity, specificity and repeatability were tested. Three days and 5 days after challenging, 88 clinical samples were detected by ECLIA and chicken embryo isolation method, and the result was compared and analyzed.【Result】The efficiency of the monoclonal antibody symbolized with [Ru（bpy）3]2＋ was 1﹕21, and the efficiency of the rabbit polyantibodies labled by biotin-streptavidin linkage system was 1﹕6. Both labeled antibodies were active in IFA and Western blotting. The detection cutoff value was 28.3, with a suspicious interval of 23.4-33.2. Negative and positive coefficients of variant were both less than 10%. The LOD（limit of detection） was 5×104 EID50. ECLIA can specially detect H9 subtype AIV, no reaction with other influenza viruses（H1, H3, H4, H5 and H6） and avian pathogens（NDV, IBV and IBDV）. The accordance rate of ECL

关 键 词：H9亚型禽流感 电化学发光免疫方法 快速检测 

分 类 号：S855.3[农业科学—临床兽医学]