夹心ELISA检测牛γ-干扰素方法的建立及应用
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- 英文论文题名：Establishment and Application of Sandwich ELISA for Detecting Bovine Interferon Gamma
- 论文作者：钱琨 ; 赵巍 ; 黄大卢 ; 邵红霞 ; 秦爱建
- 英文论文作者：QIAN Kun ; ZHAO Wei ; HUNAG Dalu ; SHAO Hongxia ; QIN Aijian ; Key Laboratory of Jiangsu Preventive Veterinary Medicine ; Yangzhou University ; Ministry of Education Key Lab for Avian Preventive Medicine ; Yangzhou University ; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses ; Yangzhou University
- 年：2016
- 作者机构：扬州大学江苏省动物预防医学重点实验室;扬州大学教育部禽类预防医学重点实验室;扬州大学江苏省动物重要疫病与人兽共患病防控协同创新中心;
- 论文关键词：牛γ-干扰素 ; 双夹心ELISA ; 应用
- 英文论文关键词：bovine interferon γ ; sandwich ELISA ; application
- 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：S858.23
- 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：7
- 文件大小：548k
- 原文格式：D
- 会议级别：全国
摘要
为建立检测牛γ-干扰素(Bo IFN-γ)的双夹心ELISA方法,将前期获得的针对Bo IFN-γ的单克隆抗体作为捕捉抗体进行夹心ELISA检测天然Bo IFN-γ,结果表明单克隆抗体Bo IFN-γ-6E5、Bo IFN-γ-3E1、Bo IFN-γ-3C2三株能捕捉到天然的牛γ干扰素。经过条件摸索和优化,最终建立了以3E1/HRP-6E5为最佳组合的检测Bo IFN-γ的夹心ELISA方法。用建立的双夹心ELISA检测方法和进口试剂盒同时对66份血样进行检测,结果表明双夹心ELISA方法和进口试剂盒检测结果的符合率达95.45%,说明该方法能有效地对牛结核病γ-干扰素进行检测。
To establish a sandwich ELISA for detection bovine interferon γ(Bo IFN-γ),three monoclonal antibodies against Bo IFN-γ were coated as capture antibody to detect natural Bo IFN-γ.The result showed that all of the three monoclonal antibodies could recognize Bo IFN-γ.After the exploration and optimization,the sandwich ELISA for detecting Bo IFN-γ were established by 3E1 as capture antibody and HRP conjugated 6E5 as detect antibody respectively.For the evaluation of this sandwich ELISA,66 serum samples from different regions were assessed both by the sandwich ELISA and by the commercial ELISA kit.The agreement between these two tests was 95.45%,which revealed that this sandwich ELISA method could effectively detect interferon γ in bovine tuberculosis disease.
To establish a sandwich ELISA for detection bovine interferon γ(Bo IFN-γ),three monoclonal antibodies against Bo IFN-γ were coated as capture antibody to detect natural Bo IFN-γ.The result showed that all of the three monoclonal antibodies could recognize Bo IFN-γ.After the exploration and optimization,the sandwich ELISA for detecting Bo IFN-γ were established by 3E1 as capture antibody and HRP conjugated 6E5 as detect antibody respectively.For the evaluation of this sandwich ELISA,66 serum samples from different regions were assessed both by the sandwich ELISA and by the commercial ELISA kit.The agreement between these two tests was 95.45%,which revealed that this sandwich ELISA method could effectively detect interferon γ in bovine tuberculosis disease.
引文
[1]Humblet,M.F.,M.L.Boschiroli,and C.Saegerman.Classification of worldwide bovine tuberculosis risk factors in cattle:a stratified approach[J].Vet Res,2009,40(5):50.
[2]Sarnaik,R.M.,et al.,Serodiagnosis of tuberculosis:assessment of kaolin agglutination test.Tuber Lung Dis,1993.74(6):p.405-6.
[3]Dalovisio,J.R.,et al.,Comparison of the amplified Mycobacterium tuberculosis(MTB)direct test,Amplicor MTB PCR,and IS6110-PCR for detection of MTB in respiratory specimens.Clin Infect Dis,1996.23(5):p.1099-106;discussion 1107-8.
[4]Sugden,E.A.,et al.,Competitive and indirect enzyme-linked immunosorbent assays for Mycobacterium bovis infections based on MPB70 and lipoarabinomannan antigens.Can J Vet Res,1997.61(1):p.8-14.
[5]Corrin,K.C.,et al.,An evaluation of the comparative tuberculin skin test for detecting tuberculosis in farmed deer.N Z Vet J,1993.41(1):p.12-20.
[6]Rothel,J.S.,et al.,A sandwich enzyme immunoassay for bovine interferon-gamma and its use for the detection of tuberculosis in cattle.Aust Vet J,1990.67(4):p.134-7.
[7]Ameni,G.,et al.,Comparison between comparative tuberculin and gamma-interferon tests for the diagnosis of bovine tuberculosis in Ethiopia.Trop Anim Health Prod,2000.32(5):p.267-76.
[8]Gonzalez Llamazares,O.R.,et al.,Field evaluation of the single intradermal cervical tuberculin test and the interferon-gamma assay for detection and eradication of bovine tuberculosis in Spain.Vet Microbiol,1999.70(1-2):p.55-66.
[9]Pollock,J.M.and S.D.Neill,Mycobacterium bovis infection and tuberculosis in cattle.Vet J,2002.163(2):p.115-27.
[10]许金俊,奶牛干扰素-γ基因的表达及其在乳房炎防治中的初步应用[D].扬州:扬州大学博士学位论文,2005.
[11]尹天燕.γ-干扰素介导牛结核快速检测方法的研究及初步应用[D].扬州:扬州大学硕士学位论文,2010.
[12]黄大卢.Bo IFN-γ基因的原核表达及其单克隆抗体的研制[D].扬州:扬州大学硕士学位论文,2012.
[13]Sattah,M.V.,et al.,Interferon-gamma release assay T-SPOT(R).TB and HIV-related tuberculosis.Int JTuberc Lung Dis,2012.16(2):p.281-2.
[14]Humblet,M.F.,M.L.Boschiroli,and C.Saegerman,Classification of worldwide bovine tuberculosis risk factors in cattle:a stratified approach.Vet Res,2009.40(5):p.50.
[15]Sabio y Garcia,J.V.,et al.,Expression of MPB83 from Mycobacterium bovis in Brucella abortus S19induces specific cellular immune response against the recombinant antigen in BALB/c mice.Microbes Infect,2010.12(14-15):p.1236-43.
[16]Sarnaik R M,et al.Serodiagnosis of tuberculosis:assessment of kaolin agglutination test[J].Tuber Lung Dis,1993,74(6):405-406.
[2]Sarnaik,R.M.,et al.,Serodiagnosis of tuberculosis:assessment of kaolin agglutination test.Tuber Lung Dis,1993.74(6):p.405-6.
[3]Dalovisio,J.R.,et al.,Comparison of the amplified Mycobacterium tuberculosis(MTB)direct test,Amplicor MTB PCR,and IS6110-PCR for detection of MTB in respiratory specimens.Clin Infect Dis,1996.23(5):p.1099-106;discussion 1107-8.
[4]Sugden,E.A.,et al.,Competitive and indirect enzyme-linked immunosorbent assays for Mycobacterium bovis infections based on MPB70 and lipoarabinomannan antigens.Can J Vet Res,1997.61(1):p.8-14.
[5]Corrin,K.C.,et al.,An evaluation of the comparative tuberculin skin test for detecting tuberculosis in farmed deer.N Z Vet J,1993.41(1):p.12-20.
[6]Rothel,J.S.,et al.,A sandwich enzyme immunoassay for bovine interferon-gamma and its use for the detection of tuberculosis in cattle.Aust Vet J,1990.67(4):p.134-7.
[7]Ameni,G.,et al.,Comparison between comparative tuberculin and gamma-interferon tests for the diagnosis of bovine tuberculosis in Ethiopia.Trop Anim Health Prod,2000.32(5):p.267-76.
[8]Gonzalez Llamazares,O.R.,et al.,Field evaluation of the single intradermal cervical tuberculin test and the interferon-gamma assay for detection and eradication of bovine tuberculosis in Spain.Vet Microbiol,1999.70(1-2):p.55-66.
[9]Pollock,J.M.and S.D.Neill,Mycobacterium bovis infection and tuberculosis in cattle.Vet J,2002.163(2):p.115-27.
[10]许金俊,奶牛干扰素-γ基因的表达及其在乳房炎防治中的初步应用[D].扬州:扬州大学博士学位论文,2005.
[11]尹天燕.γ-干扰素介导牛结核快速检测方法的研究及初步应用[D].扬州:扬州大学硕士学位论文,2010.
[12]黄大卢.Bo IFN-γ基因的原核表达及其单克隆抗体的研制[D].扬州:扬州大学硕士学位论文,2012.
[13]Sattah,M.V.,et al.,Interferon-gamma release assay T-SPOT(R).TB and HIV-related tuberculosis.Int JTuberc Lung Dis,2012.16(2):p.281-2.
[14]Humblet,M.F.,M.L.Boschiroli,and C.Saegerman,Classification of worldwide bovine tuberculosis risk factors in cattle:a stratified approach.Vet Res,2009.40(5):p.50.
[15]Sabio y Garcia,J.V.,et al.,Expression of MPB83 from Mycobacterium bovis in Brucella abortus S19induces specific cellular immune response against the recombinant antigen in BALB/c mice.Microbes Infect,2010.12(14-15):p.1236-43.
[16]Sarnaik R M,et al.Serodiagnosis of tuberculosis:assessment of kaolin agglutination test[J].Tuber Lung Dis,1993,74(6):405-406.
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