副猪嗜血杆菌抗体间接ELISA检测方法的建立及应用
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- 英文论文题名：Establishment and Application of an Indirect ELISA for Detection of Antibody against Haemophilus Parasuis
- 论文作者：马福利 ; 王丽荣 ; 董永军 ; 刘运平 ; 王秋霞 ; 梁莹莹
- 英文论文作者：MA Fuli ; WANG Lirong ; DONG Yongjun ; LIU Yunping ; WANG Qiuxia ; LIANG Yingying ; College of Animal Science ; Henan Institute of Science and Technology ; Antibody Engineering Laboratory ; Henan Institute of Science and Technology ; Xinxiang Generalization Station of Animal Husbandry Technology
- 年：2016
- 作者机构：河南科技学院动物科学学院;河南科技学院抗体工程重点实验室;新乡市畜牧技术推广站;
- 论文关键词：副猪嗜血杆菌 ; 抗体 ; 间接ELISA
- 英文论文关键词：Haemophilus parasuis ; Antibody ; Indirect ELISA
- 会议召开时间：2016-07-20
- 会议录名称：中国畜牧兽医学会兽医公共卫生学分会第五次学术研讨会论文集
- 语种：中文
- 分类号：S858.28
- 学会代码：SYGW
- 会议名称：中国畜牧兽医学会兽医公共卫生学分会第五次学术研讨会
- 会议地点：中国内蒙古通辽
- 主办单位：中国畜牧兽医学会兽医公共卫生学分会
- 学会名称：中国畜牧兽医学会兽医公共卫生学分会
- 页数：6
- 文件大小：543k
- 原文格式：D
- 会议级别：全国
摘要
采用超声裂解副猪嗜血杆菌分离株5型菌体上清作为包被抗原,初步建立副猪嗜血杆菌抗体的间接ELISA检测方法。通过棋盘滴定法筛选确定最佳反应条件:抗原包被质量浓度50 mg/L,37℃作用2 h后4℃过夜包被,血清的稀释度为1:320,抗原抗体反应时间为50 min,酶标二抗稀释度为1:12 000,作用时间为30 min,底物显色时间为15 min。经特异性、敏感性和符合性试验检测结果表明,建立的间接ELISA方法特异性和重复性良好,敏感性高,可用于副猪嗜血杆菌的检疫和流行病学调查。
An indirect ELISA coated with supernatant of sonicated Haemophilus parasuis serovar 5cell culture was established.The results of chess board titration tests showed that coating antigen at a mass concentration of 50mg/L incubated optimally at 37 ℃ for 2 h and then coated overnight at 4℃.The optimal dilution of serum and HRP-Ig G was 1:320 and 1:12 000,respectively.The reaction time of coating antigen with serum and serum with HRP-Ig G was 50 min.The time of color development reaction was 15 min.Specificity,sensitivity and compliance tests confirmed that the developed ELISA had good stability and specificity.So this assay can be used as a tool for diagnosis and quarantine of Haemophilμs parasμis.
An indirect ELISA coated with supernatant of sonicated Haemophilus parasuis serovar 5cell culture was established.The results of chess board titration tests showed that coating antigen at a mass concentration of 50mg/L incubated optimally at 37 ℃ for 2 h and then coated overnight at 4℃.The optimal dilution of serum and HRP-Ig G was 1:320 and 1:12 000,respectively.The reaction time of coating antigen with serum and serum with HRP-Ig G was 50 min.The time of color development reaction was 15 min.Specificity,sensitivity and compliance tests confirmed that the developed ELISA had good stability and specificity.So this assay can be used as a tool for diagnosis and quarantine of Haemophilμs parasμis.
引文
[1]XUE Guocong,REN Tao Research Progress on Epidemiology and Causative Agent of Haemophilus parasuis[J].China Animal Husbandry&Veterinary Medicine,2009,36(5):168-171(in Chinese with English abstract).
[2]Olvera A,Calsamiglia M,Aragon V.Genotypic diversity of Haemophilus parasuis field strains[J].Applied and Environmental Microbiology,2006,72:3984-3992.
[3]WANG Yan,JIANG Ping Development of an indirect ELISA for detection of antibodies against Haemophilus parasuis[J].Animal Husbandry&Veterinary Medicine,2006,38(3):5-7(in Chinese with English abstract).
[4]WEI Zigong,CAI Xuwang,JIN Hailin,et al.Establishment and application of an indirect hemagglutination assay for detection of antibodies against Haemophilus parasuis[J].Veterinary Science in China,2006,36(9):713-718(in Chinese with English abstract).
[5]CAO Shuze.Veterinary Microbiology and Immunology Technology[M].Beijing:Beijing Agriculture University Press,1991(in Chinese).
[6]SHI Bi,CUI Yaowen,JIA Fan,et al.Establishment of indirect haemagglutination and indirect ELISA assays for detection of antibodies against Haemophilus parasuis serotype 5[J].Veterinary Science in China,2007,37(11):964-968(in Chinese with English abstract).
[7]Hui Jin,Rui Zhou,Mingsong Kang,et al.Biofilm formation by field isolates and reference strains of Haemophilus parasuis[J].Veterinary Microbiology,2006,118(12):117-123.
[8]Oliveira S,Pijoan C.Haemophilus parasuis:new trends on diagnosis,epidemiology and control[J].Vet Microbiol,2004.99(1):1-12.
[9]CAI Xuwang,LIU Zhengfei,CHEN Huanchun,et al.Isolation and Serovar Identification of Haemophilus parasuis in China[J].Journal of Huazhong Agricultural,2005,24(1):55-58(in Chinese with English abstract).
[10]Amano H,Shibata M,Kajio N,et al.Pathologic observations of pigs intranasally inoculated with serovar 1,4and 5 of Haemophilus parasuis using immunoperoxidase method[J].Journal of Veterinary Medical Science.1994,56(4):639-644.
[11]CHU Yuefeng,GAO Pengcheng,ZHAO Ping.Establishment of KRG Method for Serotyping Haemophilus parasuis and its Application in Epidemiology Study[J].Jiangsu Journal of Agricultural Sciences,2010,26(5):999-1002(in Chinese with English abstract).
[12]Blanco I,Canals A,Evans G,et al.Differences in susceptibility to Haemophilus parasuis infection in pigs[J].Canadian Journal of Veterinary Research.2008,72:228-235.
[13]Miniats O P,S mart N L,Rosendal S.Cross protection among Haemophilusp arasuiss trainsin immunized gnotobiotic pigs[J].Can J Vet Res,1991,55(l):37-41.
[2]Olvera A,Calsamiglia M,Aragon V.Genotypic diversity of Haemophilus parasuis field strains[J].Applied and Environmental Microbiology,2006,72:3984-3992.
[3]WANG Yan,JIANG Ping Development of an indirect ELISA for detection of antibodies against Haemophilus parasuis[J].Animal Husbandry&Veterinary Medicine,2006,38(3):5-7(in Chinese with English abstract).
[4]WEI Zigong,CAI Xuwang,JIN Hailin,et al.Establishment and application of an indirect hemagglutination assay for detection of antibodies against Haemophilus parasuis[J].Veterinary Science in China,2006,36(9):713-718(in Chinese with English abstract).
[5]CAO Shuze.Veterinary Microbiology and Immunology Technology[M].Beijing:Beijing Agriculture University Press,1991(in Chinese).
[6]SHI Bi,CUI Yaowen,JIA Fan,et al.Establishment of indirect haemagglutination and indirect ELISA assays for detection of antibodies against Haemophilus parasuis serotype 5[J].Veterinary Science in China,2007,37(11):964-968(in Chinese with English abstract).
[7]Hui Jin,Rui Zhou,Mingsong Kang,et al.Biofilm formation by field isolates and reference strains of Haemophilus parasuis[J].Veterinary Microbiology,2006,118(12):117-123.
[8]Oliveira S,Pijoan C.Haemophilus parasuis:new trends on diagnosis,epidemiology and control[J].Vet Microbiol,2004.99(1):1-12.
[9]CAI Xuwang,LIU Zhengfei,CHEN Huanchun,et al.Isolation and Serovar Identification of Haemophilus parasuis in China[J].Journal of Huazhong Agricultural,2005,24(1):55-58(in Chinese with English abstract).
[10]Amano H,Shibata M,Kajio N,et al.Pathologic observations of pigs intranasally inoculated with serovar 1,4and 5 of Haemophilus parasuis using immunoperoxidase method[J].Journal of Veterinary Medical Science.1994,56(4):639-644.
[11]CHU Yuefeng,GAO Pengcheng,ZHAO Ping.Establishment of KRG Method for Serotyping Haemophilus parasuis and its Application in Epidemiology Study[J].Jiangsu Journal of Agricultural Sciences,2010,26(5):999-1002(in Chinese with English abstract).
[12]Blanco I,Canals A,Evans G,et al.Differences in susceptibility to Haemophilus parasuis infection in pigs[J].Canadian Journal of Veterinary Research.2008,72:228-235.
[13]Miniats O P,S mart N L,Rosendal S.Cross protection among Haemophilusp arasuiss trainsin immunized gnotobiotic pigs[J].Can J Vet Res,1991,55(l):37-41.