Scientia Agricultura Sinica ›› 2015, Vol. 48 ›› Issue (15): 3064-3070.doi: 10.3864/j.issn.0578-1752.2015.15.017

• SPECIAL FOCUS: THE IMPACT OF INFLUENZA VIRUSES ON HUMAN AND ANIMAL HUSBANDRY • Previous Articles     Next Articles

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

QI Wen-bao, LI Fang, LI Hua-nan, HUANG Li-hong, HE Jun, MU Guang-hui, LUO Kai-jian, LIAO Ming   

  1. College of Veterinary Medicine, South China Agricultural University/National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Guangzhou 510642
  • Received:2015-03-19 Online:2015-08-01 Published:2015-08-01

Abstract: 【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 of tripropylamine 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 ECLIA with chicken embryo isolation method was 86.4%.【Conclusion】The ECLIA could be an available tool for diagnosis and control of H9 subtype avian influenza.

Key words: H9 subtype avian influenza, Electrochemical Luminescence Immunoassay (ECLIA), quick detection method

[1]    赵国, 刘晓文, 钱忠明, 薛峰, 彭宜, 彭大新, 刘秀梵. 2002-2009 年中国华东地区家禽低致病性禽流感的病原学检测与分析. 中国农业科学, 2011, 44(1): 153-159.
Zhao G, Liu X W, Qian Z M, Xue F, Peng Y, Peng D X, Liu X F. Detection and analysis of low pathogenic avian influenza in poultry in Eastern China from 2002 to 2009. Scientia Agricultura Sinica, 2011, 44(1): 153-159. (in Chinese)
[2]    陈陆, 刘守川, 赵军, 王川庆, 王泽霖. 不同H9N2亚型禽流感病毒分离株致病力研究及HA 抗原性变异分析, 中国农业科学, 2011, 44(24): 5100-5107.
Chen L, Liu S C, Zhao J, Wang C Q, Wang Z L. Characteristics of pathogenic and HA antigenic variation of H9N2 subtybe avian influenza viruses isolated from 1998 to 2008 in China. Scientia Agricultura Sinica, 2011, 44(24): 5100-5107. (in Chinese)
[3]    Yu H, Zhou Y J, Li GX, Ma J H, Yan L P, Wang B, Yang F R, Huang M, Tong G Z. Genetic diversity of H9N2 influenza viruses from pigs in China: A potential threat to human health? Veterinary Microbiology, 2011, 149: 254-261.
[4]    Guan Y, Shortridge K F, Krauss S, Webster R G. Molecular characterization of H9N2 influenza viruses: were they the donors of the ‘‘internal’’ genes of H5N1 viruses in HongKong? Proceedings of the National Academy of Sciences of the USA, 1999, 96: 9363-9367.
[5]    Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, Chen J, Jie Z, Qiu H, Xu K, Xu X, Lu H, Zhu W, Gao Z, Xiang N, Shen Y, He Z, Gu Y, Zhang Z, Yang Y, Zhao X, Zhou L, Li X, Zou S, Zhang Y, Li X, Yang L, Guo J, Dong J, Li Q, Dong L, Zhu Y, Bai T, Wang S, Hao P, Yang W, Zhang Y, Han J, Yu H, Li D, Gao GF, Wu G, Wang Y, Yuan Z, Shu Y. Human infection with a novel avian-origin influenza A (H7N9) virus. New England Journal of Medicine, 2013, 368: 1888-1897.
[6]    Zhu H, Wang D, Kelvin D J, Li L, Zheng Z, Yoon S W, Wong S S, Farooqui A, Wang J, Banner D, Chen R, Zheng R, Zhou J, Zhang Y, Hong W, Dong W, Cai Q, Roehrl M H, Huang S S, Kelvin A A, Yao T, Zhou B, Chen X, Leung G M, Poon L L, Webster R G, Webby R J, Peiris J S, Guan Y, Shu Y. Infectivity, transmission, and pathology of human-isolated H7N9 influenza virus in ferrets and pigs. Science, 2013, 341: 183-186.
[7]    Chen H, Yuan H, Gao R, Zhang J, Wang D, Xiong Y, Fan G, Yang F, Li X, Zhou J, Zou S, Yang L, Chen T, Dong L, Bo H, Zhao X, Zhang Y, Lan Y, Bai T, Dong J, Li Q, Wang S, Zhang Y, Li H, Gong T, Shi Y, Ni X, Li J, Zhou J, Fan J, Wu J, Zhou X, Hu M, Wan J, Yang W, Li D, Wu G, Feng Z, Gao GF, Wang Y, Jin Q, Liu M, Shu Y. Clinical and epidemiological characteristics of a fatal case of avian influenza A H10N8 virus infection: a descriptive study. Lancet, 2014, 383(9918): 714-721.
[8]    Qi W, Zhou X, Shi W, Huang L, Xia W, Liu D, Li H, Chen S, Lei F, Cao L, Wu J, He F, Song W, Li Q, Li H, Liao M, Liu M. Genesis of the novel human-infecting influenza A(H10N8) virus and potential genetic diversity of the virus in poultry, China. Euro Surveillence, 2014, 19(25): 20841.
[9]    柯艳坤, 陈晓春, 齐岩, 张煜坤, 廖明, 亓文宝. 禽流感病毒检测方法的研究进展. 中国家禽, 2008, 14: 29-32.
Ke Y K, Chen X C, Qi Y, Zhang Y K, Liao M, Qi W B. Development of avian influenza virus detection method. China Poultry, 2008, 14: 29-32. (in Chinese)
[10]   Moreno A, Brocchi E, Lelli D, Gamba D, Tranquillo M, Cordioli P. Monoclonal antibody based ELISA tests to detect antibodies against neuraminidase subtypes 1, 2 and 3 of avian influenza viruses in avian sera. Vaccine, 2009, 27: 4967-4974.
[11]   胡思顺, 郑兴华, 蔡开妹, 彭伏虎, 张文泽, 李自力, 肖运才, 刘 梅, 毕丁仁. 禽流感病毒感染与疫苗免疫鉴别诊断试纸条的研制及应用. 中国农业科学, 2009, 42(2): 694-700.
Hu S S, Zheng X H, Cai K M, Peng F H, Zhang W Z, Li Z L, Xiao Y C, Liu M, Bi D R. Development of colloidal gold strip for differentiating antibody of AIV-infected flocks from vaccinated ones. Scientia Agricultura Sinica, 2009, 42(2): 694-700. (in Chinese)
[12]   Chen H T, Zhang J, Ma L N, Ma Y P, Ding Y Z, Wang M, Liu X T, Zhang Y G, Liu YS. Rapid subtyping of H9N2 influenza virus by a triple reverse transcription polymerase chain reaction. Journal of Virological Methods, 2009, 158: 58-62.
[13]   Yu H. Comparative studies of magnetic particle-based solid phase fluorogenic and electrochemiluminescent immunoassay. Journal of Immunological Methods, 1998, 218: 1-8.
[14]   Miao W, Bard A J. Electrogenerated chemiluminescence. 72. Determination of immobilized DNA and C-reactive protein on Au(111) electrodes using tris (2, 2'-bipyridyl) ruthenium(II) labels. Analytical Chemistry, 2003, 75: 5825-5834.
[15]   Dai X, Hilsen R E, Hu W G, Fulton R E. Microbead electrochemiluminescence immunoassay for detection and identification of Venezuelan equine encephalitis virus. Journal of Virological Methods, 2010, 169: 274-281.
[16]   汤巧, 吴文静, 夏永祥. 化学发光法和酶联免疫吸附法检测丙型肝炎病毒抗体的比较分析. 国际检验医学杂志, 2011, 32: 834-835.
Tang Q, Wu W J, Xia Y X. Comparison of CLIA and ELISA in the detection of anti-HCV antibody. International Journal of Laboratory Medicine, 2011, 32: 834-835. (in Chinese)
[17]   鞠毅, 邵淑丽, 于燕妮, 常爱英, 车燕妮. Elecsys2010 型全自动电化学发光免疫分析仪工作原理与故障检修. 中国医疗设备, 2008, 23(1): 96-97.
Ju Y, Shao S L, Yu Y N, Chang A Y, Che Y N. Principle and maintenance of Elecsys2010 automatic electrochemical immuno- analyzer. China Medical Equipment, 2008, 23(1): 96-97. (in Chinese)
[18]   Ramanaviciene A, German N, Kausaite-Minkstimiene A, Voronovic J, Kirlyte J, Ramanavicius A. Comparative study of surface plasmon resonance, electrochemical and electroassisted chemiluminescence methods based immunosensor for the determination of antibodies against human growth hormone.  Biosens Bioelectron, 2012, 36(1): 48-55.
[19]   Han J, Zhuo Y, Chai Y Q, Yuan Y L, Yuan R. Novel electrochemical catalysis as signal amplified strategy for label-free detection of neuron-specific enolase.  Biosens Bioelectron, 2012, 31(1): 399-405.
[20]   Li M, Li R, Li C M, Wu N. Electrochemical and optical biosensors based on nanomaterials and nanostructures: a review.  Frontiers Bioscience (Schol Ed), 2011, 3: 1308-1331.
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