流行性出血病病毒抗原捕获ELISA方法的建立
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摘要
为建立流行性出血病病毒(EHDV)病原学检测方法,本试验用纯化处理的4种血清型病毒(EHDV-2、5、6和8)混合免疫兔和豚鼠,制备高免血清。以兔抗EHDV为捕获抗体,豚鼠抗EHDV为检测抗体,羊抗豚鼠IgG-HRP为酶标抗体,建立了EHDV抗原捕获ELISA(antigen capture ELISA,AC-ELISA)方法。通过反应条件优化,获得最佳工作条件:5%马血清为稀释液,包被抗体1∶15 000稀释,检测抗体1∶20 000稀释,酶标抗体1∶15 000稀释。通过检测60份阴性样品确定临界值,P/N值≥2为阳性、P/N值<1.5为阴性;特异性试验表明该方法仅能检测出各血清型EHDV、蓝舌病病毒(BTV)、赤羽病病毒(AKAV)及中山病毒(CHUV)无交叉反应;敏感性试验表明该方法可以检出102.47±0.07 TCID50的病毒;批内和批间变异系数均<10%;用RT-PCR与该方法同时对70份参考样品进行检测,符合率为92.86%。表明本试验建立的AC-ELISA方法为EHDV抗原检测提供了一种可靠实用的技术手段。
To establish an assay for epizootic haemorrhagic disease virus(EHDV)detection,the anti-serum against EHDV were prepared with the purified EHDV-2,5,6and 8replicated in BHK-21 cell to immune the rabbit and guinea pig respectively,and then the antigen capture ELISA(ACELISA)was developed with the rabbit anti-serum as coating antibody for antigen capture and with the guinea pig anti-serum for sandwich binding antigen follow by IgG-HRP detection.The optimum conditions were achieved:5% horse serum as diluents,capture antisera was diluted for 1∶15 000,detecting antibody was diluted 1∶20 000 and the goat to Gpig IgG-HRP was diluted for1∶15 000.Using P/N value as the criteria by setting positive and negative control,serum sample with P/N ration more than or equal to 2.0were considered as positive,those less than 1.5as negative,and the rest was suspicious.The AC-ELISA was high specificity and sensitive for EHDV detection with a limit detection of 10~(2.47±0.07) TCID_(50) and no cross-reaction was found in bluetongue virus,Akabane virus and Chuzan disease virus.The intra-assay and inter-assay variation coefficient were both less 10%,and the coincidence rate with RT-PCR was 92.86%.These results demonstrated the AC-ELISA was sensitive,specific and accurate detection technology for EHDV.
To establish an assay for epizootic haemorrhagic disease virus(EHDV)detection,the anti-serum against EHDV were prepared with the purified EHDV-2,5,6and 8replicated in BHK-21 cell to immune the rabbit and guinea pig respectively,and then the antigen capture ELISA(ACELISA)was developed with the rabbit anti-serum as coating antibody for antigen capture and with the guinea pig anti-serum for sandwich binding antigen follow by IgG-HRP detection.The optimum conditions were achieved:5% horse serum as diluents,capture antisera was diluted for 1∶15 000,detecting antibody was diluted 1∶20 000 and the goat to Gpig IgG-HRP was diluted for1∶15 000.Using P/N value as the criteria by setting positive and negative control,serum sample with P/N ration more than or equal to 2.0were considered as positive,those less than 1.5as negative,and the rest was suspicious.The AC-ELISA was high specificity and sensitive for EHDV detection with a limit detection of 10~(2.47±0.07) TCID_(50) and no cross-reaction was found in bluetongue virus,Akabane virus and Chuzan disease virus.The intra-assay and inter-assay variation coefficient were both less 10%,and the coincidence rate with RT-PCR was 92.86%.These results demonstrated the AC-ELISA was sensitive,specific and accurate detection technology for EHDV.
引文
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[12]张胜男,李华春,朱建波,等.2015年内蒙古地区蓝舌病及流行性出血热流行病学调查及血清型鉴定[J].中国预防兽医学报,2016,38(12):939-943.
[13]张怡轩,林俊,曹颖颖,等.广西鹿流行性出血热病毒血清型调查及分布影响分析[J].上海畜牧兽医通讯,2016(4):19-21.
[14]刘帅,张玲,加帕尔·哈斯木,等.新疆牛群中鹿流行性出血热血清学调查[J].流行病学,2016,33(4):15-18.
[15]和东华,杨振兴,高林,等.云南省边境地区牛流行性出血病病毒血清学调查[J].云南畜牧兽医,2016(5):7-10.
[16]吕敏娜,朱建波,李娟,等.广东一株牛源流行性出血病病毒的分离鉴定[J].中国预防兽医学报,2017,39(1):67-70.
[17]曹颖颖,钟华,吴健敏.鹿流行性出血热病毒研究进展[J].中国兽医学报,2017,37(3):571-576.
[18]曹颖颖,吴健敏,林俊,等.广西首例牛源鹿流行性出血热病毒的分离鉴定[J].中国预防兽医学报,2015,37(10):746-750.
[19]SHARMA P,STALLKNECHT D,MURPHY M D,et al.Expression of interleukin-1beta and interleukin-6in white-tailed deer infected with Epizootic haemorrhagic disease virus[J].Vet Ital,2015,51(4):283-288.
[20]ARADAIB I E,MEDEROS R A,OSBURN B.Evaluation of epizootic haemorrhagic disease virus infection in sentinel calves from the SAN joaquin valley of California[J].Vet Res Commun,2005,29(5):447-451.
[21]赵传博,王丽,董波,等.猪血凝性脑脊髓炎病毒抗原捕获ELISA诊断方法的建立[J].中国兽医学报,2013,33(4):526-531.
[22]CHAND K,SANCHAY K B,DE AN-KAN,et al.Apolyclonal antibody-based sandwich ELISA for the detection of bluetongue virus in cell culture and blood of sheep infected experimentally[J].J Virol Methods,2009,160(1):189-192.
[23]SAEGERMAN C,BERKVENS D,MELLOR P S.Bluetongue epidemiology in the European Union[J].Emerg Infect Dis,2008,14(4):539-544.
[24]苗海生,李乐,廖德芳,等.蓝舌病病毒抗原捕获ELISA检测方法的建立[J].中国预防兽医学报,2015,37(3):216-219.
[2]MILLS M K,RUDER M G,NAYDUCH D,et al.Dynamics of epizootic hemorrhagic disease virus infection within the vector,Culicoides sonorensis(Diptera:Ceratopogonidae)[J].PLoS One,2017,12(11):e0188865.
[3]TEMIZEL E M,YESILBAG K,BATTEN C,et al.Epizootic hemorrhagic disease in cattle,Western Turkey[J].Emerg Infect Dis,2009,15(2):317-319.
[4]SAVINI G,AFONSO A,MELLOR P,et al.Epizootic haemorrhagic disease[J].Res Vet Sci,2011,91(1):1-17.
[5]SPEDICATO M,CARMINE I,TEODORI L,et al.Innocuity of a commercial live attenuated vaccine for epizootic hemorrhagic disease virus serotype 2in lateterm pregnant cows[J].Vaccine,2016,34(12):1430-1435.
[6]NOON T H,WESCHE S L,CAGLE D.Hemorrhagic disease in bighorn sheep in Arizona[J].J Wildl Dis,2002,38(1):172-176.
[7]OIE,Manual of diagnostic tests and vaccines for terres.OIE terrestrial manual[S].2014:chapter2.1.4B,1-11.
[8]WILSON W C,RUDER M G,KLEMENT E,et al.Genetic characterization of epizootic hemorrhagic disease virus strains isolated from cattle in Israel[J].JGen Virol,2015,96(6):1400-1410.
[9]VIAROUGE C,LANCELOT R,RIVES G,et al.Identification of bluetongue virus and epizootic hemorrhagic disease virus serotypes in French Guiana in2011and 2012[J].Vet Microbiol,2014,174(1/2):78-85.
[10]SHIRAFUJI H,KATO T,YAMAKAWA M,et al.Characterization of genome segments 2,3and 6of epizootic hemorrhagic disease virus strains isolated in Japan in 1985-2013:Identification of their serotypes and geographical genetic types[J].Infect Genet Evol,2017,53(5):38-46.
[11]MAAN N S,MAAN S,POTGIETER A C,et al.Development of real-time RT-PCR assays for detection and typing of epizootic haemorrhagic disease virus[J].Transbound Emerg Dis,2017,64(6):1120-1132.
[12]张胜男,李华春,朱建波,等.2015年内蒙古地区蓝舌病及流行性出血热流行病学调查及血清型鉴定[J].中国预防兽医学报,2016,38(12):939-943.
[13]张怡轩,林俊,曹颖颖,等.广西鹿流行性出血热病毒血清型调查及分布影响分析[J].上海畜牧兽医通讯,2016(4):19-21.
[14]刘帅,张玲,加帕尔·哈斯木,等.新疆牛群中鹿流行性出血热血清学调查[J].流行病学,2016,33(4):15-18.
[15]和东华,杨振兴,高林,等.云南省边境地区牛流行性出血病病毒血清学调查[J].云南畜牧兽医,2016(5):7-10.
[16]吕敏娜,朱建波,李娟,等.广东一株牛源流行性出血病病毒的分离鉴定[J].中国预防兽医学报,2017,39(1):67-70.
[17]曹颖颖,钟华,吴健敏.鹿流行性出血热病毒研究进展[J].中国兽医学报,2017,37(3):571-576.
[18]曹颖颖,吴健敏,林俊,等.广西首例牛源鹿流行性出血热病毒的分离鉴定[J].中国预防兽医学报,2015,37(10):746-750.
[19]SHARMA P,STALLKNECHT D,MURPHY M D,et al.Expression of interleukin-1beta and interleukin-6in white-tailed deer infected with Epizootic haemorrhagic disease virus[J].Vet Ital,2015,51(4):283-288.
[20]ARADAIB I E,MEDEROS R A,OSBURN B.Evaluation of epizootic haemorrhagic disease virus infection in sentinel calves from the SAN joaquin valley of California[J].Vet Res Commun,2005,29(5):447-451.
[21]赵传博,王丽,董波,等.猪血凝性脑脊髓炎病毒抗原捕获ELISA诊断方法的建立[J].中国兽医学报,2013,33(4):526-531.
[22]CHAND K,SANCHAY K B,DE AN-KAN,et al.Apolyclonal antibody-based sandwich ELISA for the detection of bluetongue virus in cell culture and blood of sheep infected experimentally[J].J Virol Methods,2009,160(1):189-192.
[23]SAEGERMAN C,BERKVENS D,MELLOR P S.Bluetongue epidemiology in the European Union[J].Emerg Infect Dis,2008,14(4):539-544.
[24]苗海生,李乐,廖德芳,等.蓝舌病病毒抗原捕获ELISA检测方法的建立[J].中国预防兽医学报,2015,37(3):216-219.