猪戊型肝炎病毒血清抗体间接ELISA检测方法的建立与应用
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摘要
由于猪戊型肝炎病毒(hepatitis E virus,HEV)可跨种属感染人,因此,猪群中猪HEV感染的监测对于该病的防控和公共卫生都具有重要的意义。本试验旨在建立一种检测猪HEV抗体的间接ELISA方法,以原核表达纯化截短的猪HEV衣壳蛋白为包被抗原,优化条件,确定最佳试验条件,并分析该方法的敏感性、特异性、重复性,以及与商品化试剂盒的符合率,评价其临床应用价值。SDS-PAGE和Western blot结果证明抗原蛋白获得正确表达与纯化,并且具有良好的反应原性,可以作为后续建立ELISA方法的包被抗原。通过优化ELISA条件,最终确定抗原最佳包被量为200ng·孔-1,血清最佳稀释度为1∶200,包被缓冲液为碳酸盐缓冲液(pH 9.6),封闭液为2.5%的脱脂奶粉,阴阳性判定的临界值为OD450nm≥0.296。该间接ELISA方法的特异性和敏感性良好,板内和板间变异系数均小于10%,与北京万泰商品化试剂盒符合率为91.2%。临床应用表明,该方法可应用于猪HEV感染猪后抗体消长以及临床猪血清抗体的检测。因此,本研究建立的猪HEV抗体间接ELISA检测方法具有较好的敏感性和特异性,准确率较高,该方法适用于临床猪血清样品的检测和猪HEV感染血清流行病学的调查。
Swine hepatitis E virus(HEV)can infect human by cross-species transmission.Therefore,it is important for surveillance by detection of swine HEV infection in pig herds.In this study,an indirect ELISA for detecting anti-swine HEV IgG antibodies was developed using a truncated swine HEV ORF2 protein expressed in a prokaryotic system as the coating antigen.The procedures of indirect ELISA were also optimized.The sensitivity,specificity,reproducibility,and comparisons with the commercial kit were analyzed.In addition,the application of indirect ELISA for detecting anti-swine HEV IgG antibodies in the field serum samples was also evaluated.SDS-PAGE and Western blot analysis showed that the truncated ORF2 protein was successfully expressed and reacted with the positive pig sera for swine HEV antibodies.The optimalamount of the coating antigen was determined at 200 ng per well and the dilution of sera was 1:200.The optimal coating and blocking buffers were carbonate buffer(pH=9.6)and 2.5% dry milk buffer,respectively.The cut-off value of the indirect ELISA was determined as OD450 nm≥0.296.The sensitivity and specificity were 100% and 1:6 400,respectively.Both intra-and inter-assay variabilities were less than 10%.The confidence between the indirect ELISA and the Wantai commercial kit was 91.2%.The developed indirect ELISA can also be used to detect antiswine HEV IgG antibodies in the sequential sera from the pigs challenged with swine HEV and the clinical pig sera.Collectively,the developed indirect ELISA have good sensitivity,specificity and accuracy,and can be used for detecting anti-swine HEV IgG antibodies in pig sera from the field and ser-epidemiology investigation for swine HEV infection in pig herds.
Swine hepatitis E virus(HEV)can infect human by cross-species transmission.Therefore,it is important for surveillance by detection of swine HEV infection in pig herds.In this study,an indirect ELISA for detecting anti-swine HEV IgG antibodies was developed using a truncated swine HEV ORF2 protein expressed in a prokaryotic system as the coating antigen.The procedures of indirect ELISA were also optimized.The sensitivity,specificity,reproducibility,and comparisons with the commercial kit were analyzed.In addition,the application of indirect ELISA for detecting anti-swine HEV IgG antibodies in the field serum samples was also evaluated.SDS-PAGE and Western blot analysis showed that the truncated ORF2 protein was successfully expressed and reacted with the positive pig sera for swine HEV antibodies.The optimalamount of the coating antigen was determined at 200 ng per well and the dilution of sera was 1:200.The optimal coating and blocking buffers were carbonate buffer(pH=9.6)and 2.5% dry milk buffer,respectively.The cut-off value of the indirect ELISA was determined as OD450 nm≥0.296.The sensitivity and specificity were 100% and 1:6 400,respectively.Both intra-and inter-assay variabilities were less than 10%.The confidence between the indirect ELISA and the Wantai commercial kit was 91.2%.The developed indirect ELISA can also be used to detect antiswine HEV IgG antibodies in the sequential sera from the pigs challenged with swine HEV and the clinical pig sera.Collectively,the developed indirect ELISA have good sensitivity,specificity and accuracy,and can be used for detecting anti-swine HEV IgG antibodies in pig sera from the field and ser-epidemiology investigation for swine HEV infection in pig herds.
引文
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[2] WHO.Hepatitis E[EB/OL].(2017-07-03)[2018-07-04]. http://www.who.int/news-room/factsheets/detail/hepatitis-e.
[3] GUO Q S,YAN Q,XIONG J H,et al.Prevalence of hepatitis E virus in Chinese blood donors[J].J Clin Microbiol,2010,48(1):317-318.
[4] MA L,SUN P,LIN F Z,et al.Prevalence of hepatitis E virus in Chinese blood donors[J].J Int Med Res,2015,43(2):257-262.
[5] MENG X J,PURCELL R H,HALBUR P G,et al.A novel virus in swine is closely related to the human hepatitis E virus[J].Proc Natl Acad Sci U S A,1997,94(18):9860-9865.
[6] YUGO D M,MENG X J.Hepatitis E virus:Foodborne,waterborne and zoonotic transmission[J].Int J Environ Res Public Health,2013,10(10):4507-4533.
[7] LIU P,LI L J,WANG L,et al.Phylogenetic analysis of 626hepatitis E virus(HEV)isolates from humans and animals in China(1986-2011)showing genotype diversity and zoonotic transmission[J].Infect Genet Evol,2012,12(2):428-434.
[8] HUANG F F,HAQSHENAS G,GUENETTE D K,et al.Detection by reverse transcription-PCR and genetic characterization of field isolates of swine hepatitis E virus from pigs in different geographic regions of the United States[J].J Clin Microbiol,2002,40(4):1326-1332.
[9]戎广亚,乔小江,张建宗,等.以合成肽作抗原的酶联免疫吸附法诊断戊型肝炎病毒感染[J].中华医学检验杂志,1994,17(2):75-77.RONG G Y,QIAO X J,ZHANG J Z,et al.Serodiagnosis of hepatitis E virus infection by an ELISA based on synthetic antigens[J].Chinese Journal of Medical Laboratory Sciences,1994,17(2):75-77.(in Chinese)
[10]刘启文,李震,赵凯,等.猪戊型肝炎ELISA诊断试剂盒的研制及应用[J].畜牧兽医学报,2010,41(11):1435-1441.LIU Q W,LI Z,ZHAO K,et al.Preparation and application of swine hepatitis E virus diagnostic ELISA kit[J].Acta Veterinaria et Zootechnica Sinica,2010,41(11):1435-1441.(in Chinese)
[11] AVELLON A,MORAGO L,DEL CARMEN M G G,et al.Comparative sensitivity of commercial tests for hepatitis E genotype 3virus antibody detection[J].J Med Virol,2015,87(11):1934-1939.
[12] KAMAR N,IZOPET J,PAVIO N,et al.Hepatitis E virus infection[J/OL].Nat Rev Dis Primers,2017,3:17086[2018-07-04].http://www.nature.com/articles/nrdp201786.
[13] SMITH D B,SIMMONDS P,JAMEEL S,et al.Consensus proposals for classification of the family Hepeviridae[J].J Gen Virol,2015,96(5):1191-1192.
[14]赵宇军,许冬梅,朱元茂,等.猪戊型肝炎病毒结构蛋白片段的表达及其在ELISA中的初步应用[J].畜牧兽医学报,2006,37(11):1198-1201.ZHAO Y J,XU D M,ZHU Y M,et al.Expression of swine hepatitis E Virus ORF2and development of ELISA based on the expressed protein[J].Acta Veterinaria et Zootechnica Sinica,2006,37(11):1198-1201.(in Chinese)
[15]郭志,陈飞,刘水芳,等.猪戊型肝炎病毒ORF2基因不同抗原区域的原核表达及鉴定[J].中国兽医科学,2014,44(2):159-164.GUO Z,CHEN F,LIU S F,et al.Prokaryotic expression and antigenic analysis of the different antigen regions of ORF2gene of swine hepatitis E virus[J].Chinese Veterinary Science,2014,44(2):159-164.(in Chinese)
[16]闻晓波,王密,冉旭华,等.猪戊型肝炎病毒ORF2主要抗原表位区间接ELISA方法的初步建立[J].中国畜牧兽医,2010,37(5):99-102.WEN X B,WANG M,RAN X H,et al.Development of indirect ELISA with major epitope domain of ORF2of sHEV[J].China Animal Husbandry&Veterinary Medicine,2010,37(5):99-102.(in Chinese)
[17] ZHAO Q,SYED S F,ZHOU E M.Antigenic properties of avian hepatitis E virus capsid protein[J].Vet Microbiol,2015,108(1-2):10-14.
[18]帅江冰,张晓峰,徐晶靓,等.2005-2008年浙江省生猪主产区猪戊型肝炎血清流行病学调查[J].畜牧兽医学报,2009,40(7):1037-1042.SHUAI J B,ZHANG X F,XU J L,et al.Seroprevalence of hepatitis E virus infections in pigs from Zhejiang Province during 2005-2008[J].Acta Veterinaria et Zootechnica Sinica,2009,40(7):1037-1042.(in Chinese)
[19]张厚勇,陈东升,张懿,等.规模化猪场戊型肝炎病毒感染状况的调查与分析[J].畜牧兽医学报,2010,41(10):1268-1273.ZHANG H Y,CHEN D S,ZHANG Y,et al.Epidemiological survey of hepatitis E virus in industralized swine herds[J].Acta Veterinaria et Zootechnica Sinica,2010,41(10):1268-1273.(in Chinese)
[20]张中锋,金宁一,朱光泽,等.长春地区人与猪戊型肝炎病毒分子流行病学及部分基因序列分析[J].中国人兽共患病学报,2006,22(10):941-945.ZHANG Z F,JIN N Y,ZHU G Z,et al.Partial nucleotide sequencing and molecular epidemiology of hepatitis E viruses from swine and patients with hepatitis E infection in Changchun of Jilin province[J].Chinese Journal of Zoonoses,2006,22(10):941-945.(in Chinese)
[21]黄芬,禹文海,马天武,等.云南某农村猪戊型肝炎病毒分子流行病学调查[J].中国人兽共患病学报,2012,28(3):274-277.HUANG F,YU W H,MA T W,et al.Molecular epidemiology of swine HEV in a village of Yunnan[J].Chinese Journal of Zoonoses,2012,28(3):274-277.(in Chinese)
[2] WHO.Hepatitis E[EB/OL].(2017-07-03)[2018-07-04]. http://www.who.int/news-room/factsheets/detail/hepatitis-e.
[3] GUO Q S,YAN Q,XIONG J H,et al.Prevalence of hepatitis E virus in Chinese blood donors[J].J Clin Microbiol,2010,48(1):317-318.
[4] MA L,SUN P,LIN F Z,et al.Prevalence of hepatitis E virus in Chinese blood donors[J].J Int Med Res,2015,43(2):257-262.
[5] MENG X J,PURCELL R H,HALBUR P G,et al.A novel virus in swine is closely related to the human hepatitis E virus[J].Proc Natl Acad Sci U S A,1997,94(18):9860-9865.
[6] YUGO D M,MENG X J.Hepatitis E virus:Foodborne,waterborne and zoonotic transmission[J].Int J Environ Res Public Health,2013,10(10):4507-4533.
[7] LIU P,LI L J,WANG L,et al.Phylogenetic analysis of 626hepatitis E virus(HEV)isolates from humans and animals in China(1986-2011)showing genotype diversity and zoonotic transmission[J].Infect Genet Evol,2012,12(2):428-434.
[8] HUANG F F,HAQSHENAS G,GUENETTE D K,et al.Detection by reverse transcription-PCR and genetic characterization of field isolates of swine hepatitis E virus from pigs in different geographic regions of the United States[J].J Clin Microbiol,2002,40(4):1326-1332.
[9]戎广亚,乔小江,张建宗,等.以合成肽作抗原的酶联免疫吸附法诊断戊型肝炎病毒感染[J].中华医学检验杂志,1994,17(2):75-77.RONG G Y,QIAO X J,ZHANG J Z,et al.Serodiagnosis of hepatitis E virus infection by an ELISA based on synthetic antigens[J].Chinese Journal of Medical Laboratory Sciences,1994,17(2):75-77.(in Chinese)
[10]刘启文,李震,赵凯,等.猪戊型肝炎ELISA诊断试剂盒的研制及应用[J].畜牧兽医学报,2010,41(11):1435-1441.LIU Q W,LI Z,ZHAO K,et al.Preparation and application of swine hepatitis E virus diagnostic ELISA kit[J].Acta Veterinaria et Zootechnica Sinica,2010,41(11):1435-1441.(in Chinese)
[11] AVELLON A,MORAGO L,DEL CARMEN M G G,et al.Comparative sensitivity of commercial tests for hepatitis E genotype 3virus antibody detection[J].J Med Virol,2015,87(11):1934-1939.
[12] KAMAR N,IZOPET J,PAVIO N,et al.Hepatitis E virus infection[J/OL].Nat Rev Dis Primers,2017,3:17086[2018-07-04].http://www.nature.com/articles/nrdp201786.
[13] SMITH D B,SIMMONDS P,JAMEEL S,et al.Consensus proposals for classification of the family Hepeviridae[J].J Gen Virol,2015,96(5):1191-1192.
[14]赵宇军,许冬梅,朱元茂,等.猪戊型肝炎病毒结构蛋白片段的表达及其在ELISA中的初步应用[J].畜牧兽医学报,2006,37(11):1198-1201.ZHAO Y J,XU D M,ZHU Y M,et al.Expression of swine hepatitis E Virus ORF2and development of ELISA based on the expressed protein[J].Acta Veterinaria et Zootechnica Sinica,2006,37(11):1198-1201.(in Chinese)
[15]郭志,陈飞,刘水芳,等.猪戊型肝炎病毒ORF2基因不同抗原区域的原核表达及鉴定[J].中国兽医科学,2014,44(2):159-164.GUO Z,CHEN F,LIU S F,et al.Prokaryotic expression and antigenic analysis of the different antigen regions of ORF2gene of swine hepatitis E virus[J].Chinese Veterinary Science,2014,44(2):159-164.(in Chinese)
[16]闻晓波,王密,冉旭华,等.猪戊型肝炎病毒ORF2主要抗原表位区间接ELISA方法的初步建立[J].中国畜牧兽医,2010,37(5):99-102.WEN X B,WANG M,RAN X H,et al.Development of indirect ELISA with major epitope domain of ORF2of sHEV[J].China Animal Husbandry&Veterinary Medicine,2010,37(5):99-102.(in Chinese)
[17] ZHAO Q,SYED S F,ZHOU E M.Antigenic properties of avian hepatitis E virus capsid protein[J].Vet Microbiol,2015,108(1-2):10-14.
[18]帅江冰,张晓峰,徐晶靓,等.2005-2008年浙江省生猪主产区猪戊型肝炎血清流行病学调查[J].畜牧兽医学报,2009,40(7):1037-1042.SHUAI J B,ZHANG X F,XU J L,et al.Seroprevalence of hepatitis E virus infections in pigs from Zhejiang Province during 2005-2008[J].Acta Veterinaria et Zootechnica Sinica,2009,40(7):1037-1042.(in Chinese)
[19]张厚勇,陈东升,张懿,等.规模化猪场戊型肝炎病毒感染状况的调查与分析[J].畜牧兽医学报,2010,41(10):1268-1273.ZHANG H Y,CHEN D S,ZHANG Y,et al.Epidemiological survey of hepatitis E virus in industralized swine herds[J].Acta Veterinaria et Zootechnica Sinica,2010,41(10):1268-1273.(in Chinese)
[20]张中锋,金宁一,朱光泽,等.长春地区人与猪戊型肝炎病毒分子流行病学及部分基因序列分析[J].中国人兽共患病学报,2006,22(10):941-945.ZHANG Z F,JIN N Y,ZHU G Z,et al.Partial nucleotide sequencing and molecular epidemiology of hepatitis E viruses from swine and patients with hepatitis E infection in Changchun of Jilin province[J].Chinese Journal of Zoonoses,2006,22(10):941-945.(in Chinese)
[21]黄芬,禹文海,马天武,等.云南某农村猪戊型肝炎病毒分子流行病学调查[J].中国人兽共患病学报,2012,28(3):274-277.HUANG F,YU W H,MA T W,et al.Molecular epidemiology of swine HEV in a village of Yunnan[J].Chinese Journal of Zoonoses,2012,28(3):274-277.(in Chinese)