肠道病毒68型新型中和试验方法的建立及其初步应用
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摘要
针对肠道病毒68型(EV-D68)建立一种新型快速、高效的中和试验方法,并初步评价其在EV-D68流行病学研究中的应用价值。以灭活EV-D68病毒免疫Balb/c小鼠,利用杂交瘤技术制备其单克隆抗体;筛选反应性好、特异性强的抗EV-D68单克隆抗体作为检测抗体,基于酶联免疫斑点检测技术(ELISPOT)建立EV-D68高效中和试验方法;进一步比较该方法与传统中和试验方法 Nt-CPE的一致性,并使用该方法对临床血清样本进行检测。共获得10株分泌抗EV-D68单克隆抗体的杂交瘤细胞株,选取一株性能最优的15C5进行生产纯化,鉴定其为IgG3亚型;以15C5为检测抗体,建立了快速检测EV-D68中和抗体的Nt-ELISPOT方法;与Nt-CPE方法比较,Nt-ELISPOT方法将检测时间由5~7d缩短至1d以内,并且两种方法检测结果一致性良好;应用所建立的Nt-ELISPOT方法对厦门地区146份人群血清样本进行检测,显示血清样本EV-D68中和抗体阳性率为98.6%(144/146),提示厦门地区可能存在过EV-D68的流行。本研究基于ELISPOT建立的新型EV-D68中和试验方法快速可靠,适合通量检测,可以应用于EV-D68中和抗体水平的血清学调查,为EV-D68感染的防控工作提供帮助。
We wished to establish a novel,rapid and efficient neutralization test for enterovirus D68(EVD68)and evaluate its application in seroepidemiological studies.Balb/c mice were immunized with inactivated EV-D68.Monoclonal antibodies against EV-D68 were prepared by the hybridoma method.Anti-EVD68 monoclonal antibodies with high affinity and specificity were selected as detection antibodies for the establishment of a rapid neutralization test for EV-D68 using the enzyme-linked immunospot(ELISPOT)assay.Also,the consistency between the established method and conventional one,neutralization test based on the inhibition of cytopathic effects(Nt-CPE),was investigated.Finally,the established method was evaluated with clinical serum samples.Ten hybridoma cell lines secreting monoclonal antibodies against EVD68 were obtained,and the best of them,15C5,was selected for the production and characterization of monoclonal antibodies.Results showed that 15C5 was an immunoglobulin(Ig)G3subclass.A rapid method for the detection of neutralizing antibodies against EV-D68,Nt-ELISPOT,was established using 15C5 as the detection antibody.Nt-ELISPOT could shorten the detection time substantially from 5~7days in conventional Nt-CPE to <1day,and there was good consistency between the two methods.The established Nt-ELISPOT was applied to 146 clinical serum samples from Xiamen,China,and the positive detection rate was 98.6%(144/146),suggesting that EV-D68 infection might be prevalent in Xiamen.The established novel neutralization test for EV-D68 based on ELISPOT was rapid and reliable.This method could be used to detect EV-D68 neutralizing antibodies in seroepidemiological studies,thereby providing support for the prevention and control of EV-D68 infection.
We wished to establish a novel,rapid and efficient neutralization test for enterovirus D68(EVD68)and evaluate its application in seroepidemiological studies.Balb/c mice were immunized with inactivated EV-D68.Monoclonal antibodies against EV-D68 were prepared by the hybridoma method.Anti-EVD68 monoclonal antibodies with high affinity and specificity were selected as detection antibodies for the establishment of a rapid neutralization test for EV-D68 using the enzyme-linked immunospot(ELISPOT)assay.Also,the consistency between the established method and conventional one,neutralization test based on the inhibition of cytopathic effects(Nt-CPE),was investigated.Finally,the established method was evaluated with clinical serum samples.Ten hybridoma cell lines secreting monoclonal antibodies against EVD68 were obtained,and the best of them,15C5,was selected for the production and characterization of monoclonal antibodies.Results showed that 15C5 was an immunoglobulin(Ig)G3subclass.A rapid method for the detection of neutralizing antibodies against EV-D68,Nt-ELISPOT,was established using 15C5 as the detection antibody.Nt-ELISPOT could shorten the detection time substantially from 5~7days in conventional Nt-CPE to <1day,and there was good consistency between the two methods.The established Nt-ELISPOT was applied to 146 clinical serum samples from Xiamen,China,and the positive detection rate was 98.6%(144/146),suggesting that EV-D68 infection might be prevalent in Xiamen.The established novel neutralization test for EV-D68 based on ELISPOT was rapid and reliable.This method could be used to detect EV-D68 neutralizing antibodies in seroepidemiological studies,thereby providing support for the prevention and control of EV-D68 infection.
引文
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[11]李姝璇,杨立生,侯汪衡,赵欢,万俊凯,陈梦媛,何水珍,程通,夏宁邵.埃可病毒25型新型中和实验方法的建立与应用[J].生物技术通讯,2016,27(1):52-57.
[12]Zielinska E,Liu D,Wu H Y,Quiroz J,Rappaport R,Yang D P.Development of an improved microneutralization assay for respiratory syncytial virus by automated plaque counting using imaging analysis[J].Virol J,2005,2:84.
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[14]Liu L,Wen K,Li J,Hu D,Huang Y,Qiu L,Cai J,Che X.Comparison of plaque-and enzyme-linked immunospot-based assays to measure the neutralizing activities of monoclonal antibodies specific to domain III of dengue virus envelope protein[J].Clin Vaccine Immunol,2012,19(1):73-78.
[15]Wang W,Cheng T,Ma K,Xia D,Wang Y,Liu J,Du H,Shih J W K,Zhang J,Zhao Q,Xia N.Development of a novel baculovirus titration method using the Enzyme-linked immunosorbent spot(ELISPOT)assay[J].J Virol Methods,2013,188(1-2):114-120.
[16]Nakane P K,Kawaoi A.Peroxidase-labeled antibody.A new method of conjugation[J].J Histochem Cytochem,1974,22(12):1084-1091.
[17]Zhuge J,Vail E,Bush J L,Singelakis L,Huang W,Nolan S M,Haas J P,Engel H,Della Posta M,Yoon E C,Fallon J T,Wang G.Evaluation of a real-time reverse transcription-PCR assay for detection of enterovirus D68in clinical samples from an outbreak in new york state in 2014[J].J Clin Microbiol,2015,53(6):1915-1920.
[18]Wylie T N,Wylie K M,Buller R S,Cannella M,Storch G A.Development and evaluation of an enterovirus D68 real-time reverse transcriptase PCR assay[J]。J Clin Microbiol,2015,53(8):2641-2647.
[19]Kung S H,Wang S F,Huang C W,Hsu C C,Liu H F,Yang J Y.Genetic and antigenic analyses of enterovirus 71isolates in Taiwan during 1998-2005[J].Clin Microbiol Infect,2007,13(8):782-787.
[20]Chang L Y,King C C,Hsu K H,Ning H C,Tsao K C,Li C C,Huang Y C,Shih S R,Chiou S T,Chen P Y,Chang H J,Lin T Y.Risk factors of enterovirus 71infection and associated hand,foot,and mouth disease/herpangina in children during an epidemic in Taiwan[J/OL].Pediatrics,2002,109(6):e88.
[2]Piralla A,Girello A,Grignani M,Gozalo-Marguello M,Marchi A,Marseglia G,Baldanti F.Phylogenetic characterization of enterovirus 68strains in patients with respiratory syndromes in Italy[J].J Med Virol,2014,86(9):1590-1593.
[3]Meijer A,van der Sanden S,Snijders B E,JaramilloGutierrez G,Bont L,van der Ent C K,Overduin P,Jenny S L,Jusic E,van der Avoort H G,Smith G J,Donker G A,Koopmans M P.Emergence and epidemic occurrence of enterovirus 68 respiratory infections in The Netherlands in 2010[J].Virology,2012,423(1):49-57.
[4]Lu Q B,Wo Y,Wang H Y,Wei M T,Zhang L,Yang H,Liu E M,Li T Y,Zhao Z T,Liu W,Cao W C.Detection of enterovirus 68as one of the commonest types of enterovirus found in patients with acute respiratory tract infection in China[J].J Med Microbiol,2014,63(Pt 3):408-414.
[5]Holm-Hansen C C,Midgley S E,Fischer T K.Global emergence of enterovirus D68:a systematic review[J/OL].Lancet Infect Dis,2016,16(5):e64-e75.
[6]Poelman R,Schuffenecker I,Van Leer-Buter C,Josset L,Niesters H G,Lina B.European surveillance for enterovirus D68during the emerging North-American outbreak in 2014[J].J Clin Virol,2015,711-719.
[7]Midgley C M,Watson J T,Nix W A,Curns A T,Rogers S L,Brown B A,Conover C,Dominguez S R,Feikin D R,Gray S,Hassan F,Hoferka S,Jackson M A,Johnson D,Leshem E,Miller L,Nichols J B,Nyquist A C,Obringer E,Patel A,Patel M,Rha B,Schneider E,Schuster J E,Selvarangan R,Seward J F,Turabelidze G,Oberste M S,Pallansch M A,Gerber S I.Severe respiratory illness associated with a nationwide outbreak of enterovirus D68in the USA(2014):a descriptive epidemiological investigation[J].Lancet Respir Med,2015,3(11):879-887.
[8]Hou W,Yang L,He D,Zheng J,Xu L,Liu J,Liu Y,Zhao H,Ye X,Cheng T,Xia N.Development of a coxsackievirus A16neutralization test based on the enzymelinked immunospot assay[J].J Virol Methods,2015,215-216:56-60.
[9]Huang M L,Chiang P S,Luo S T,Liou G Y,Lee M S.Development of a high-throughput assay for measuring serum neutralizing antibody against enterovirus 71[J].J Virol Methods,2010,165(1):42-45.
[10]Yang L,He D,Tang M,Li Z,Liu C,Xu L,Chen Y,Du H,Zhao Q,Zhang J,Cheng T,Xia N.Development of an enzyme-linked immunosorbent spot assay to measure serum-neutralizing antibodies against coxsackievirus B3[J].Clin Vaccine Immunol,2014,21(3):312-320.
[11]李姝璇,杨立生,侯汪衡,赵欢,万俊凯,陈梦媛,何水珍,程通,夏宁邵.埃可病毒25型新型中和实验方法的建立与应用[J].生物技术通讯,2016,27(1):52-57.
[12]Zielinska E,Liu D,Wu H Y,Quiroz J,Rappaport R,Yang D P.Development of an improved microneutralization assay for respiratory syncytial virus by automated plaque counting using imaging analysis[J].Virol J,2005,2:84.
[13]Li T,Lin H,Yu L,Xue M,Ge S,Zhao Q,Zhang J,Xia N.Development of an enzyme-linked immunospot assay for determination of rotavirus infectivity[J].J Virol Methods,2014,209:7-209:14.
[14]Liu L,Wen K,Li J,Hu D,Huang Y,Qiu L,Cai J,Che X.Comparison of plaque-and enzyme-linked immunospot-based assays to measure the neutralizing activities of monoclonal antibodies specific to domain III of dengue virus envelope protein[J].Clin Vaccine Immunol,2012,19(1):73-78.
[15]Wang W,Cheng T,Ma K,Xia D,Wang Y,Liu J,Du H,Shih J W K,Zhang J,Zhao Q,Xia N.Development of a novel baculovirus titration method using the Enzyme-linked immunosorbent spot(ELISPOT)assay[J].J Virol Methods,2013,188(1-2):114-120.
[16]Nakane P K,Kawaoi A.Peroxidase-labeled antibody.A new method of conjugation[J].J Histochem Cytochem,1974,22(12):1084-1091.
[17]Zhuge J,Vail E,Bush J L,Singelakis L,Huang W,Nolan S M,Haas J P,Engel H,Della Posta M,Yoon E C,Fallon J T,Wang G.Evaluation of a real-time reverse transcription-PCR assay for detection of enterovirus D68in clinical samples from an outbreak in new york state in 2014[J].J Clin Microbiol,2015,53(6):1915-1920.
[18]Wylie T N,Wylie K M,Buller R S,Cannella M,Storch G A.Development and evaluation of an enterovirus D68 real-time reverse transcriptase PCR assay[J]。J Clin Microbiol,2015,53(8):2641-2647.
[19]Kung S H,Wang S F,Huang C W,Hsu C C,Liu H F,Yang J Y.Genetic and antigenic analyses of enterovirus 71isolates in Taiwan during 1998-2005[J].Clin Microbiol Infect,2007,13(8):782-787.
[20]Chang L Y,King C C,Hsu K H,Ning H C,Tsao K C,Li C C,Huang Y C,Shih S R,Chiou S T,Chen P Y,Chang H J,Lin T Y.Risk factors of enterovirus 71infection and associated hand,foot,and mouth disease/herpangina in children during an epidemic in Taiwan[J/OL].Pediatrics,2002,109(6):e88.