埃博拉病毒抗体间接血凝检测方法的建立
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摘要
以纯化后的埃博拉病毒(Ebola virus,EBOV)糖蛋白(GP)作为抗原,致敏醛化的绵羊红细胞,进行最佳反应条件优化,建立间接血凝抗体检测方法。以EBOV高免马血清、纯化的IgG和精制免疫球蛋白F(ab’)_2为待检样品进行检测,并将检测结果与假病毒中和试验检测结果相比较。结果显示,该方法可特异性检测EBOV抗体,与马尔堡病毒、裂谷热病毒等的阳性血清均不发生反应;与假病毒中和试验测得的抗体效价增长规律相一致。结果表明,本试验成功建立了EBOV抗体间接血凝检测方法,该方法安全、简便、快捷,为EBOV疫苗免疫后的效果评价提供了又一新的检测方法。
To establish an indirect hemagglutination assay(IHA) for Ebola virus(EBOV) antibody,the purified EBOV glycoprotein(GP) was used as antigen to sensitize the hydroformylation of sheep red blood cells for optimal reaction conditions.The EBOV hyperimmune horse serum,purified IgG and purified immunoglobulin F(ab')_2 were tested,and the results were compared with that of the pseudovirus neutralization test.The results showed that this method can specifically detect EBOV antibody,and do not react to positive sera of Marburg virus,Rift Valley fever virus,etc.;the results were consistent with that of the pseudovirus neutralization test.In this study,an indirect hemagglutination assay for EBOV antibody was successfully established.This method is safe,simple and fast,which provided a new detection method for evaluation of the immune efficacy after EBOV vaccine inoculation.
To establish an indirect hemagglutination assay(IHA) for Ebola virus(EBOV) antibody,the purified EBOV glycoprotein(GP) was used as antigen to sensitize the hydroformylation of sheep red blood cells for optimal reaction conditions.The EBOV hyperimmune horse serum,purified IgG and purified immunoglobulin F(ab')_2 were tested,and the results were compared with that of the pseudovirus neutralization test.The results showed that this method can specifically detect EBOV antibody,and do not react to positive sera of Marburg virus,Rift Valley fever virus,etc.;the results were consistent with that of the pseudovirus neutralization test.In this study,an indirect hemagglutination assay for EBOV antibody was successfully established.This method is safe,simple and fast,which provided a new detection method for evaluation of the immune efficacy after EBOV vaccine inoculation.
引文
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[13] FELDMANN H,VOLCHKOV V E,VOLCHKOVA V A,et al.Biosynthesis and role of filoviral glycoproteins[J].J Gen Virol,2001,82(2):2839-2848.
[14] 夏咸柱,许基龙,胡敬尧,等.用反向间接血凝检测乙脑病毒抗原的试验[J].中国兽医学报,1981,1(3):38-40.
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[16] 曹增国,王化磊,盖微微,等.埃博拉病毒抗体间接ELISA检测方法的建立及应用[J].畜牧兽医学报,2016,47(3):615-619.
[17] MARTIN P,LAUPLAND K B,FROST E H,et al.Laboratory diagnosis of Ebola virus disease[J].Intensive Care Med,2015,41(5):895-898.
[18] 张守发,张国宏,王浩然,等.应用间接血凝试验诊断猪附红细胞体病[J].中国兽医杂志,2004,40(8):17-18.
[19] 何光志,田维毅,简昌友,等.应用间接血凝试验(IHA)诊断猪蛔虫病[J].贵州农业科学,2010,38(8):153-155.
[20] 刘忠伟,周碧君,易见刚,等.应用正向间接血凝试验检测山羊痘抗体[J].山地农业生物学报,2005,24(5):397-400.
[21] 魏子贡,蔡旭旺,金梅林,等.副猪嗜血杆菌抗体间接血凝检测方法的建立及应用[J].中国兽医科学,2006,36(9):713-718.
[22] 万永虎,杨桃梅,张德著,等.醛化红细胞应用的概述[J].黑龙江畜牧兽医,2015(11):79-81.
[23] 张慧辉,刘丽艳,陈永耀.不同醛化方法制备的鸡红细胞对新城疫病毒的血凝对比试验[J].河南畜牧兽医(综合版),2007,28(7):7-8.
[24] 赵伟,胡业勤,薛红刚,等.醛化鹅红细胞的制备及其在百日咳血凝活性测定中的应用[J].中国生物制品学杂志,2012,25(11):1535-1537.
[25] YU J M,DE VLAS S J,JIANG Q W,et al.Comparison of the Kato-Katz technique,hatching test and indirect hemagglutination assay (IHA) for the diagnosis of Schistosoma japonicum infection in China[J].Parasitol Int,2007,56(1):45-49.
[26] 北京医学院微生物学教研组.实验免疫学[M].北京:人民卫生出版社,1980:396-415.
[2] GEISBERT T W,JAHRLING P B.Exotic emerging viral diseases:progress and challenges[J].Nat Med,2004,10(Suppl):110-121.
[3] World Health Organization.Ebola virus disease situation report[EB/OL].[2016-06-10].http://apps.who.int/iris/bitstream/10665/208883/1/ebolasitrep_10-Jun2016_eng.pdf?ua=1.
[4] LEDGERWOOD J E,DEZURE A D,STANLEY D A,et al.Chimpanzee adenovirus vector Ebola vaccine-preliminary report[R].doi:10.1056/NEJMoa1410863.the VRC 207 Study Team.2014.
[5] XU C P,WANG H L,JIN H L,et al.Visual detection of Ebola virus using reverse transcription loopmediated isothermal amplification combined with nucleic acid strip detection[J].Arch Virol,2016,161:1125-1133.
[6] TOWNER J S,ROLLIN P E,BAUSCH D G,et al.Rapid diagnosis of Ebola hemorrhagic fever by reverse transcription-PCR in an outbreak setting and assessment of patient viral load as a predictor of outcome[J].J Virol,2004,78(8):4330-4341.
[7] VOLCHKOVA V A,FELDMANN H,KLENK H D,et al.The nonstructural small glycoprotein s GP of Ebola virus is secreted as an antiparallel-orientated homodimer[J].Virology,1998,250(2):408-414.
[8] SANCHEZ A,ROLLIN P E.Complete genome sequence of an Ebola virus (Sudan species) responsible for a 2000 outbreak of human disease in Uganda[J].Virus Res,2005,113(1):16-25.
[9] YANG Z,DELGADO R,XU L,et al.Distinct cellular interactions of secreted and transmembrane Ebola virus glycoproteins[J].Science,1998,279(5353):1034-1037.
[10] CARETTE J E,RAABEN M,WONG A C,et al.Ebola virus entry requires the cholesterol transporter Niemann-Pick C1[J].Nature,2011,477(7364):340.
[11] VOLCHKOV V E,VOLCHKOVA V A,MUHLBERGER E,et al.Recovery of infectious Ebola virus from complementary DNA:RNA editing of the GP gene and viral cytotoxicity[J].Science,2001,291(5510):1965-1969.
[12] SIMMONS G,WOOL-LEWIS R J,BARIBAUD F,et al.Ebola virus glycoproteins induce global surface protein down-modulation and loss of cell adherence[J].J Virol,2002,76(5):2518-2528.
[13] FELDMANN H,VOLCHKOV V E,VOLCHKOVA V A,et al.Biosynthesis and role of filoviral glycoproteins[J].J Gen Virol,2001,82(2):2839-2848.
[14] 夏咸柱,许基龙,胡敬尧,等.用反向间接血凝检测乙脑病毒抗原的试验[J].中国兽医学报,1981,1(3):38-40.
[15] 刘宝全,孙惠兰,李俊宝,等.兽医免疫学实验指导[M].上海:上海科学技术出版社,1985:1-19.
[16] 曹增国,王化磊,盖微微,等.埃博拉病毒抗体间接ELISA检测方法的建立及应用[J].畜牧兽医学报,2016,47(3):615-619.
[17] MARTIN P,LAUPLAND K B,FROST E H,et al.Laboratory diagnosis of Ebola virus disease[J].Intensive Care Med,2015,41(5):895-898.
[18] 张守发,张国宏,王浩然,等.应用间接血凝试验诊断猪附红细胞体病[J].中国兽医杂志,2004,40(8):17-18.
[19] 何光志,田维毅,简昌友,等.应用间接血凝试验(IHA)诊断猪蛔虫病[J].贵州农业科学,2010,38(8):153-155.
[20] 刘忠伟,周碧君,易见刚,等.应用正向间接血凝试验检测山羊痘抗体[J].山地农业生物学报,2005,24(5):397-400.
[21] 魏子贡,蔡旭旺,金梅林,等.副猪嗜血杆菌抗体间接血凝检测方法的建立及应用[J].中国兽医科学,2006,36(9):713-718.
[22] 万永虎,杨桃梅,张德著,等.醛化红细胞应用的概述[J].黑龙江畜牧兽医,2015(11):79-81.
[23] 张慧辉,刘丽艳,陈永耀.不同醛化方法制备的鸡红细胞对新城疫病毒的血凝对比试验[J].河南畜牧兽医(综合版),2007,28(7):7-8.
[24] 赵伟,胡业勤,薛红刚,等.醛化鹅红细胞的制备及其在百日咳血凝活性测定中的应用[J].中国生物制品学杂志,2012,25(11):1535-1537.
[25] YU J M,DE VLAS S J,JIANG Q W,et al.Comparison of the Kato-Katz technique,hatching test and indirect hemagglutination assay (IHA) for the diagnosis of Schistosoma japonicum infection in China[J].Parasitol Int,2007,56(1):45-49.
[26] 北京医学院微生物学教研组.实验免疫学[M].北京:人民卫生出版社,1980:396-415.