猪流行性乙型脑炎病毒prM-E抗原捕获ELISA检测方法的建立
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摘要
为建立猪流行性乙型脑炎病毒(JEV)的检测方法,本研究以pr M-E结构蛋白特异性单克隆抗体(MAb)5B10和5E7分别作为捕获抗体和辣根过氧化酶标记的检测抗体,对反应条件进行优化,建立了抗原捕获ELISA(AC-ELISA)方法。该方法与其它常见猪病病原无交叉反应,特异性强。该方法可以检出1.25×105 pfu/m L的病毒和48.44 ng/m L pr M-E重组蛋白,最佳线性检测范围为0.05μg/m L~1.00μg/m L,线性相关系数(R2)为0.996。组装的试剂盒批内和批间重复性试验的变异系数均小于10%,具有良好的重复性。试剂盒4℃保存12个月稳定。该研究为JEV亚单位疫苗的研制与生产提供了技术支持。
To develop a method for detecting porcine Japanese encephalitis virus(JEV), an antigen capture ELISA(AC-ELISA) was established with monoclonal antibody(MAb) 5B10 as capture antibody and horseradish peroxidase conjugated MAb 5E7 as detection antibody. The AC-ELISA was specificity which had no cross-reactivity with other related pathogens of swine diseases. The detection limit was 1.25 ×105pfu/m L for JEV and 48.44 ng/m L for viral pr M-E protein expressed by mammalian cell lines. The linear quantitative range was 0.05 μg/m L to 1.00 μg/m L, and the liner correlation(R2) was 0.996. The coefficients of variation intra-assay and inter-assay were less than 10%. The AC-ELISA kit was validity for 12 months stored at 4℃. The established AC-ELISA kit provided technical support to the development and production of JEV subunit vaccine.
To develop a method for detecting porcine Japanese encephalitis virus(JEV), an antigen capture ELISA(AC-ELISA) was established with monoclonal antibody(MAb) 5B10 as capture antibody and horseradish peroxidase conjugated MAb 5E7 as detection antibody. The AC-ELISA was specificity which had no cross-reactivity with other related pathogens of swine diseases. The detection limit was 1.25 ×105pfu/m L for JEV and 48.44 ng/m L for viral pr M-E protein expressed by mammalian cell lines. The linear quantitative range was 0.05 μg/m L to 1.00 μg/m L, and the liner correlation(R2) was 0.996. The coefficients of variation intra-assay and inter-assay were less than 10%. The AC-ELISA kit was validity for 12 months stored at 4℃. The established AC-ELISA kit provided technical support to the development and production of JEV subunit vaccine.
引文
[1]Hanna J N,Ritchie S A,Phillips D A,et al.An outbreak of Japanese encephalitis in the Torres Strait,Australia,1995[J].Med J Aust,1996,165(5):256-260.
[2]Hanna J N,Ritchie S A,Phillips D A,et al.Japanese encephalitis in north Queensland,Australia,1998[J].Med J Aust,1999,170(11):533-536.
[3]Mackenzie J S,Johansen C A,Ritchie S A,et al.Japanese encephalitis as an emergingvirus:the emergence and spread of Japanese encephalitis virus in Australasia[J].Curr Top Microbiol Immunol,2002,267:49-73.
[4]Van den Hurk A F,Ritchie S A,Mackenzie J S.Ecology and geographical expansion of Japanese encephalitis virus[J].Annu Rev Entomol,2009,54:17-35.
[5]Simpson D I,Smith C E,Marshall T F,et al.Arbovirus infections in Sarawak:the role of the domestic pig[J].Roy Soc Trop Med Hyg,^1976,70(1):66-72.。
[6]Unni S K,Ruzek D,Chhatbar C,et al.Japanese encephalitis virus:from genome to infectome[J].Microbes Infect,2011,13(4):312-321.
[7]Campbell G L,Hills S L,Fischer M,et al.Estimated global incidence of Japanese encephalitis:a systematic review[J].Bull World Health Organ,2011,89(10):766-774.
[8]赵凡凡,游卫云,王晓杜,等.猪日本乙型脑炎检测技术研究进展[J].中国畜牧兽医,2011,38(10):174-179.
[9]Hua Rong-hong,Li Ye-nan,Chen Zhen-shi,et al.Generation and characterization of a new mammaliancell line continuously expressing virus-like particles of Japanese encephalitis virus for a subunit vaccine candidate[J].BMC Biotechnol,2014,14(1):62-68.
[10]于丹,李静,李军,等.ELISA定量检测乙型脑炎病毒抗原[J].临床检验杂志,2009,27(6):440-441.
[11]张国强,冯素英,沈淑芹.乙型脑炎病毒抗原定量检测试剂的研制[J].中国生物制品学杂志,2006,5:511-513.
[12]Kumar J S,Parida M,Rao P V.Monoclonal antibody based antigen captureimmunoassay for detection of circulating non structural protein NS1:Implications for early diagnosis of japanese encephalitis virus infection[J].J Virol,2011,83(6):1063-1070.
[13]Mei L,Wu P,Ye J,et al.Development and application of an antigen capture ELISA assayfor diagnosis of Japanese encephalitis virus in swine,human and mosquito[J].Virol J,2012,9(1):4-10.
[14]Konishi E,Shoda M,Ajiro N,et al.Development and evaluation of an enzyme-linked immunosorbent assay for quantifying antibodies to Japanese encephalitis virus nonstructural 1 protein to detect subclinical infections in vaccinated horses[J].J Clin Microbiol,2004,42(11):5087-5093.
[2]Hanna J N,Ritchie S A,Phillips D A,et al.Japanese encephalitis in north Queensland,Australia,1998[J].Med J Aust,1999,170(11):533-536.
[3]Mackenzie J S,Johansen C A,Ritchie S A,et al.Japanese encephalitis as an emergingvirus:the emergence and spread of Japanese encephalitis virus in Australasia[J].Curr Top Microbiol Immunol,2002,267:49-73.
[4]Van den Hurk A F,Ritchie S A,Mackenzie J S.Ecology and geographical expansion of Japanese encephalitis virus[J].Annu Rev Entomol,2009,54:17-35.
[5]Simpson D I,Smith C E,Marshall T F,et al.Arbovirus infections in Sarawak:the role of the domestic pig[J].Roy Soc Trop Med Hyg,^1976,70(1):66-72.。
[6]Unni S K,Ruzek D,Chhatbar C,et al.Japanese encephalitis virus:from genome to infectome[J].Microbes Infect,2011,13(4):312-321.
[7]Campbell G L,Hills S L,Fischer M,et al.Estimated global incidence of Japanese encephalitis:a systematic review[J].Bull World Health Organ,2011,89(10):766-774.
[8]赵凡凡,游卫云,王晓杜,等.猪日本乙型脑炎检测技术研究进展[J].中国畜牧兽医,2011,38(10):174-179.
[9]Hua Rong-hong,Li Ye-nan,Chen Zhen-shi,et al.Generation and characterization of a new mammaliancell line continuously expressing virus-like particles of Japanese encephalitis virus for a subunit vaccine candidate[J].BMC Biotechnol,2014,14(1):62-68.
[10]于丹,李静,李军,等.ELISA定量检测乙型脑炎病毒抗原[J].临床检验杂志,2009,27(6):440-441.
[11]张国强,冯素英,沈淑芹.乙型脑炎病毒抗原定量检测试剂的研制[J].中国生物制品学杂志,2006,5:511-513.
[12]Kumar J S,Parida M,Rao P V.Monoclonal antibody based antigen captureimmunoassay for detection of circulating non structural protein NS1:Implications for early diagnosis of japanese encephalitis virus infection[J].J Virol,2011,83(6):1063-1070.
[13]Mei L,Wu P,Ye J,et al.Development and application of an antigen capture ELISA assayfor diagnosis of Japanese encephalitis virus in swine,human and mosquito[J].Virol J,2012,9(1):4-10.
[14]Konishi E,Shoda M,Ajiro N,et al.Development and evaluation of an enzyme-linked immunosorbent assay for quantifying antibodies to Japanese encephalitis virus nonstructural 1 protein to detect subclinical infections in vaccinated horses[J].J Clin Microbiol,2004,42(11):5087-5093.