| I群禽腺病毒间接33K-ELISA检测方法的建立 |
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| 引用本文： | 邓显文,罗思思,谢芝勋,谢丽基,刘加波,谢志勤,庞耀珊,范晴.I群禽腺病毒间接33K-ELISA检测方法的建立[J].南方农业学报,2012,43(9):1405-1408. |
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| 作者姓名： | 邓显文 罗思思 谢芝勋 谢丽基 刘加波 谢志勤 庞耀珊 范晴 |
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| 作者单位： | 广西兽医研究所/广西畜禽疫苗新技术重点实验室 |
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| 基金项目： | 国家百千万人才工程人选专项资金项目(945200603);广西特聘专家专项经费资助项目(2011B020);广西科技攻关项目(桂科攻0815009-3-6,2010GXNSFA013090,2010GXNSFA013089) |
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| 摘 要： | 【目的】建立一种鉴别I群禽腺病毒（FAVI）灭活苗免疫和野毒感染的检测方法，为FAVI的防控与净化提供技术支撑。【方法】以FAVI 33K重组蛋白为包被抗原，筛选和优化间接ELISA的条件，建立FAVI抗体的间接33K-ELISA检测方法，检验其特异性、敏感性和重复性，并鉴别检测FAVI活病毒感染血清和灭活苗免疫血清各50份。【结果】33K重组抗原最佳包被浓度6.0 μg/mL，包被条件为37℃孵育1 h后4℃过夜，最佳封闭液为5%脱脂奶，封闭时间1.0 h；待检血清稀释度1∶100，抗原抗体最佳作用时间1.0 h；酶标二抗最佳稀释度1∶3000，作用时间45 min。优化后的间接33K-ELISA的阴、阳性临界值为0.316。其特异性、敏感性、重复性试验及鉴别检测结果表明，优化后的间接33K-ELISA具有特异性强、灵敏度高、重复性好等优点，且能成功鉴别FAVI感染与灭活疫苗接种。【结论】间接33K-ELISA操作简便、特异性强、灵敏度高、重复性好、适于批量检测，可有效鉴别FAVI感染和灭活苗免疫接种，利于挑选出FAVI感染动物，为FAVI的防控与净化提供了新思路和新方法。
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| 关 键 词： | I群禽腺病毒 33K蛋白 间接ELISA 优化 |
Establishment of indirect ELISA for detection of antibodies against fowl adenovirus group Ⅰ(FAVI) |
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| DENG Xian-wen,LUO Si-si,XIE Zhi-xun,XIE Li-ji,LIU Jia-bo,XIE Zhi-qin,PANG Yao-shan,FAN Qing.Establishment of indirect ELISA for detection of antibodies against fowl adenovirus group Ⅰ(FAVI)[J].Journal of Southern Agriculture,2012,43(9):1405-1408. |
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| Authors: | DENG Xian-wen LUO Si-si XIE Zhi-xun XIE Li-ji LIU Jia-bo XIE Zhi-qin PANG Yao-shan FAN Qing |
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| Institution: | (Guangxi Veterinary Research Institute,Guangxi Key Laboratory of Animal Vaccines and New Technologies,Nanning 530001,China) |
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| Abstract: | Objective]The aim of the present experiment was to establish a detection method to differentiate infected animals from inactive-and-vaccinated animals in the fowl adenovirus group I(FAVI) in order to provide technical support for controlling and purifying FAVI.Method]Using nonstructural protein 33K recombinant protein as coating antigen,indirect 33K-ELISA was constructed through screening and optimizing reaction conditions;its specificity,sensitivity,and repeatability were tested and used to detect fifty infected and fifty inactive-and-vaccinated sera,respectively.Result]The optimal concentration of coating antigen was 6.0 μg/ml,and the optimal coating condition was incubating at 37℃ for 1h and then coating overnight at 4℃.The optimal blocking buffer was 5% skimmed milk with 1.0 h blocking time.The optimal dilution of serum vs.enzyme labeled antibody were 1:100 and 1:3000,respectively.The optimal reaction time for coating antigen with serum and enzyme labeled antibody were 60 min and 45 min,respectively.With critical value of 0.316,the optimized 33K-ELISA was proved to be of favorable specificity,high sensibility,and excellent stability;it could also successfully distinguish the infected animals from inactive-and-vaccinated animals of FAVI.Conclusion]Indirect 33K-ELISA,which can differentiate infected ones from inactive-and-vaccinated animals,is straightforward,specific,sensitive,stable,and suitable for batch detection.Due to its multiple positive traits,indirect 33K-ELISA introduced new viewpoints and methods for FAVI control and purification. |
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| Keywords: | fowl adenovirus group Ⅰ 33K protein indirect ELISA optimization |
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