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H5亚型禽流感病毒反应原性差异及ELISA
检测方法的研究
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宋建领1，张文东2，张富强3，李作生3，冯子良3，吕粤2，岳亮2，王金萍1，
胡媛媛1，张应国2，范泉水3，邱薇3
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(1.云南省热带亚热带动物病毒病重点实验室,云南 昆明650224;2.云南省动物疫病预防控制中心,
云南 昆明650051;3.成都军区疾病预防控制中心,云南 昆明650032)
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摘要：
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在获得禽流感病毒多克隆抗体及H5亚型特异性单克隆抗体的基础上，建立了H5亚型特异性抗原捕捉ELISA检测方法。通过分析不同单克隆抗体与不同禽流感毒株的反应性差异，筛选高特异性和高敏感性的H5亚型单克隆抗体。优化反应条件，确定包被抗体、检测抗体及酶标抗体的最佳工作浓度，进行敏感性、特异性、重复性及稳定性分析，并与斑点ELISA、H5N1多重RT-PCR或病毒分离鉴定的结果相比较，同时使用该方法对野外样品进行了检测。结果表明，该方法敏感、特异，具有良好的重复性和稳定性，可用于检测临床样品、鸡胚培养物及细胞培养物中的H5亚型禽流感病毒。 |
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关键词：
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禽流感病毒；H5亚型特异性；抗原捕捉ELISA |
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中图分类号：S 852.659.5 文献标识码：
A 文章编号：1673-4696(2007)10-0839-06 |
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Analysis of viral reactogenicity difference and development of H5
subtype-specific ELISA for the detection of avian influenza virus |
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SONG Jian-ling1,ZHANG Wen-dong2,ZHANG
Fu-qiang3,LI Zuo-sheng3,FENG Zi-liang3,L
Yue2,
YUE Liang2,WANG Jin-ping1,HU Yuan-yuan1,ZHANG
Ying-guo2,FAN Quan-shui3,QIU Wei3 |
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(1.Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory,Kunming 650224,China;2.Yunnan
Province Centre for Prevention and Control of Animal Diseases,Kunming 650051,China;3.Chengdu
Military Region’s Centre for Prevention and Control of Diseases,Kunming 650032,China ) |
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Abstract: Based on the polyclonal antibody and H5 subtype-specific monoclonal antibody against avian influenza virus(AIV),an antigen capture ELISA(AC-ELISA) specific to H5 subtype AIV was developed.The H5 subtype-specific monoclonal antibody with high specificity and sensitivity was selected by analysis of reactivity difference between different monoclonal antibodies or different isolates of AIV.The reaction condition and the working concentrations of coating antibody,detection antibody and enzyme-labeled antibody were optimized,and the assay’s sensitivity,specificity,repeatability,stability were analyzed and compared with those of Dot-ELISA，H5N1 multiplex RT-PCR and the virus isolation and identification.The results indicated that the developed AC-ELISA was sensitive,specific,and of good repeatability and stabi-lity for detection of H5 subtype AIV in clinical samples,embryo and cell culture tissues. |
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Key words: avian influenza virus;H5 subtype-specific;antigen capture ELISA |
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