牛病毒性腹泻病毒抗原捕获ELISA方法的建立
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摘要
用兔抗牛病毒性腹泻病毒(BVDV)多抗作为包被抗体,BVDV NS3单克隆抗体作为捕获抗体,建立了检测BVDV抗原的捕获ELISA方法,对各项反应条件进行优化,最终获得最佳工作条件为兔多抗1∶1 600稀释包被,NS3单抗1∶2 000稀释,酶标抗体工作浓度为1∶4 000稀释。特异性和敏感性试验结果表明,该方法对牛轮状病毒、牛传染性鼻气管炎病毒、牛分支杆菌无特异性交叉反应,其最低可检测7.9×103个TCID50的病毒量,与RT-PCR方法的相比较,符合率为100%。所建立的BVDV抗原捕获ELISA方法快速、特异、敏感,可用于BVDV抗原的检测。
A antigen capture enzyme-linked immunosorbent assay(ELISA)method was developed to detect antigen of bovine virus diarrhea virus(BVDV)using mouse monoclonal antibody against NS3 protein of BVDV as capture antibody and polyclonal antiserum(rabbit serum against BVDV)as coating antibody.The optimum conditions were achieved:coating antibody was diluted for 1∶1 600,the mouse monoclonal antibody was diluted for 1∶2 000 and the enzyme-label antibody was diluted for 1∶4 000.BRV,IBRV and MB were detected by the Ag-capture ELISA.The results showed that there was no cross reaction with BVDV.The method has a minimum detection concentration 7.9×103 TCID50.The result of positive detection by Ag-capture ELISA was consistent with RT-PCR.The result showed that the Ag-capture ELISA was highly rapid,specific and sensitive,and could be used for detecting BVDV antigen.
A antigen capture enzyme-linked immunosorbent assay(ELISA)method was developed to detect antigen of bovine virus diarrhea virus(BVDV)using mouse monoclonal antibody against NS3 protein of BVDV as capture antibody and polyclonal antiserum(rabbit serum against BVDV)as coating antibody.The optimum conditions were achieved:coating antibody was diluted for 1∶1 600,the mouse monoclonal antibody was diluted for 1∶2 000 and the enzyme-label antibody was diluted for 1∶4 000.BRV,IBRV and MB were detected by the Ag-capture ELISA.The results showed that there was no cross reaction with BVDV.The method has a minimum detection concentration 7.9×103 TCID50.The result of positive detection by Ag-capture ELISA was consistent with RT-PCR.The result showed that the Ag-capture ELISA was highly rapid,specific and sensitive,and could be used for detecting BVDV antigen.
引文
[1]殷震,刘景华.动物病毒学报[M].2版.北京:科学出版社,1997:562-571.
[2]Meyling A,Houe H,Jensen A M.Epidemiology of bovine virus diarrhoea virus[J].Rev Sci Tech,1990(9):75-93.
[3]邱昌庆,郭慧琛,程淑敏,等.安徽、江苏、广西部分地区水牛牛病毒性腹泻/粘膜病血清学监测[J].中国预防兽医学报,2000,22(6):453-454.
[4]BVDV virus causes heavy losses in a Scottish cattle herd[J].Vet Rec,2007,160(9):281-284.
[5]Negron M,Raizman E A,Pogranichniy R,et al.Survey on management practices related to the prevention and control of bovine viral diarrhea virus on dairy farms in Indiana,United States[J].Prev Vet Med,2011,99(2-4):130-135.
[6]Walz P H,Grooms D L,Passler T,et al.Control of bovine viral diarrhea virus in ruminants[J].J Vet Intern Med,2010,24(3):476-486.
[7]Qing F,Zhixun X,Liji X,et al.A reverse transcription loopmediated isothermal amplification method for rapid detection of bovine viral diarrhea virus[J].J Viro Meth,2012,186(1-2):43-48.
[8]范晴,谢芝勋,刘加波,等.牛病毒性腹泻病毒实时荧光定量RT-PCR检测方法的建立[J].动物医学进展,2010,30(10):10-14.
[9]范晴,谢芝勋,刘加波.牛病毒性腹泻病毒三种核酸检测方法的比较[J].中国兽医科学,2012,42(3):294-298.
[10]SN/T1905-2007.牛病毒性腹泻/粘膜病反转录聚合酶链反应操作规程[S].
[11]王树成,林建辉,刘宏,等.用MDBK传代细胞繁殖BVD毒株的研究[J].中国动物检疫,1995,12(4):6-8.
[12]Dirk D,Edward J D,Michael J,et al.Mapping of two antigenic domains on the NS3protein of the pestivirus bovine viral diarrhea virus[J].Vet Microbiol,2005,108(1-2):1322.
[13]Marzocca M P,Seki C,Giambiagi S M,et al.Truncated E2of bovine viral diarrhea virus(BVDV)expressed in Drosophila melanogaster cells:a candidate antigen for a BVDV ELISA[J].J Virol Meth,2007,144(1-2):49-56.
[14]Ferrer F,Zoth S C,Calamante G,et al.Induction of virusneutralizing antibodies by immunization with Rachiplusia nu per os infected with a recombinant baculovirus expressing the E2glycoprotein of bovine viral diarrhea virus[J].J Virol Meth,2007,146(1-2):424-427.
[2]Meyling A,Houe H,Jensen A M.Epidemiology of bovine virus diarrhoea virus[J].Rev Sci Tech,1990(9):75-93.
[3]邱昌庆,郭慧琛,程淑敏,等.安徽、江苏、广西部分地区水牛牛病毒性腹泻/粘膜病血清学监测[J].中国预防兽医学报,2000,22(6):453-454.
[4]BVDV virus causes heavy losses in a Scottish cattle herd[J].Vet Rec,2007,160(9):281-284.
[5]Negron M,Raizman E A,Pogranichniy R,et al.Survey on management practices related to the prevention and control of bovine viral diarrhea virus on dairy farms in Indiana,United States[J].Prev Vet Med,2011,99(2-4):130-135.
[6]Walz P H,Grooms D L,Passler T,et al.Control of bovine viral diarrhea virus in ruminants[J].J Vet Intern Med,2010,24(3):476-486.
[7]Qing F,Zhixun X,Liji X,et al.A reverse transcription loopmediated isothermal amplification method for rapid detection of bovine viral diarrhea virus[J].J Viro Meth,2012,186(1-2):43-48.
[8]范晴,谢芝勋,刘加波,等.牛病毒性腹泻病毒实时荧光定量RT-PCR检测方法的建立[J].动物医学进展,2010,30(10):10-14.
[9]范晴,谢芝勋,刘加波.牛病毒性腹泻病毒三种核酸检测方法的比较[J].中国兽医科学,2012,42(3):294-298.
[10]SN/T1905-2007.牛病毒性腹泻/粘膜病反转录聚合酶链反应操作规程[S].
[11]王树成,林建辉,刘宏,等.用MDBK传代细胞繁殖BVD毒株的研究[J].中国动物检疫,1995,12(4):6-8.
[12]Dirk D,Edward J D,Michael J,et al.Mapping of two antigenic domains on the NS3protein of the pestivirus bovine viral diarrhea virus[J].Vet Microbiol,2005,108(1-2):1322.
[13]Marzocca M P,Seki C,Giambiagi S M,et al.Truncated E2of bovine viral diarrhea virus(BVDV)expressed in Drosophila melanogaster cells:a candidate antigen for a BVDV ELISA[J].J Virol Meth,2007,144(1-2):49-56.
[14]Ferrer F,Zoth S C,Calamante G,et al.Induction of virusneutralizing antibodies by immunization with Rachiplusia nu per os infected with a recombinant baculovirus expressing the E2glycoprotein of bovine viral diarrhea virus[J].J Virol Meth,2007,146(1-2):424-427.