IPMA筛选猪繁殖与呼吸综合征病毒单克隆抗体方法研究
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摘要
为了得到一种能够高通量检测细胞培养物的方法,并将其用于筛选特异性强、敏感度高的抗猪繁殖与呼吸综合征病毒(PRRSV)单克隆抗体。用每孔能感染约100个细胞的病毒量接种单层覆盖96孔板的Marc-145细胞,12 h后用含3%H_2O_2的甲醇固定细胞,以制备免疫过氧化物酶单层细胞试验(IPMA)反应板。以100μL/孔的量将融合后继续培养10 d的杂交瘤细胞的培养上清加入至IPMA反应板,以辣根过氧化物酶标记的羊抗鼠IgG-HRP作为二抗,以3-氨基-9-乙基咔唑(AEC)作为显色底物,于倒置显微镜下进行观察。结果表明,共筛选出1D1、7G8等39份PRRSV单克隆抗体,这39份单抗能够使PRRSV中高致病毒株HN07-1和经典毒株BJ-4感染的Marc-145细胞被特异性染色,而对猪瘟病毒、猪伪狂犬病毒、猪流行性腹泻病毒感染的Marc-145细胞无交叉染色。因此,构建的IPMA方法能够敏感、准确地捕捉到PRRSV单克隆抗体。
To develop a high-throughput cell culture detection method for screening specific and sensitive monoclonal antibodies against Porcine reproductive and respiratory syndrome virus(PRRSV). A single layer of Marc-145 cells covered with 96-well plates was inoculated with a virus amount of about 100 cells infected per well. After 12 h,cells were fixed with 3% H_2O_2 in methanol to prepare an immunoperoxidase monolayer cell assay(Immunoperoxidase monolayer assay,IPMA)reaction plate. The culture supernatant of the hybridoma cells cultured for 10 days after the fusion was added to the IPMA reaction plate in an amount of 100 μL/well,and horseradish peroxidase-labeled goat anti-mouse IgG-HRP was used as the secondary antibody and 3-amino-9-ethylcarbazole(AEC)was used as a chromogenic substrate,then observed under an inverted microscope. The results showed that 39 PRRSV monoclonal antibodies,such as 1 D1 and 7 G8,were screened,and these 39 monoclonal antibodies were able to specifically stain Marc-145 cells infected with high-viral strain HN07-1 and classical strain BJ-4 PRRSV. There was no cross-staining of Marc-145 cells infected with Classical swine fever virus,Pseudorabies virus,and Porcine epidemic diarrhea virus. Therefore,this IPMA method constructed by the study can capture PRRSV monoclonal antibodies sensitively and accurately.
To develop a high-throughput cell culture detection method for screening specific and sensitive monoclonal antibodies against Porcine reproductive and respiratory syndrome virus(PRRSV). A single layer of Marc-145 cells covered with 96-well plates was inoculated with a virus amount of about 100 cells infected per well. After 12 h,cells were fixed with 3% H_2O_2 in methanol to prepare an immunoperoxidase monolayer cell assay(Immunoperoxidase monolayer assay,IPMA)reaction plate. The culture supernatant of the hybridoma cells cultured for 10 days after the fusion was added to the IPMA reaction plate in an amount of 100 μL/well,and horseradish peroxidase-labeled goat anti-mouse IgG-HRP was used as the secondary antibody and 3-amino-9-ethylcarbazole(AEC)was used as a chromogenic substrate,then observed under an inverted microscope. The results showed that 39 PRRSV monoclonal antibodies,such as 1 D1 and 7 G8,were screened,and these 39 monoclonal antibodies were able to specifically stain Marc-145 cells infected with high-viral strain HN07-1 and classical strain BJ-4 PRRSV. There was no cross-staining of Marc-145 cells infected with Classical swine fever virus,Pseudorabies virus,and Porcine epidemic diarrhea virus. Therefore,this IPMA method constructed by the study can capture PRRSV monoclonal antibodies sensitively and accurately.
引文
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[14] Dea S,Gagnon C A,Mardassi H,Pirzadeh B,Rogan D.Current knowledge on the structural proteins of porcine reproductive and respiratory syndrome(PRRS)virus:comparison of the North American and European Isolates[J].Arch Virol,2000,145(4):659-688.
[15] Guo Z,Chen X X,Li R,Qiao S,Zhang G .The prevalent status and genetic diversity of Porcine reproductive and respiratory syndrome virus in China:A molecular epidemiological perspective[J].Virol J,2018,15:2.doi:10.1186/s12985-017-0910-6.
[16] Wang F X,Yang Y,Liu X,He M H,Liu Y,Sun N,Zhu H W,Ren J Q,Wu H,Wen Y J.Development of a monoclonal antibody for differentiating Porcine reproductive and respiratory syndrome virus and identification of a novel non-structural protein 2 epitope peptide[J].Virus Disease,2017,28(4):408-415.doi:10.1007/s13337-017-0400-x.
[17] Plagemann P G.Epitope specificity of monoclonal antibodies to the N-protein of Porcine reproductive and respiratory syndrome virus determined by ELISA with synthetic peptides[J].Vet Immunol Immunopathol,2005,104(1/2):59-68.doi:10.1016/j.vetimm.2004.10.004.
[18] 胡美茹,孙英勋,董家新,于鸣,舒翠玲.间接ELISA法用于细胞抗原特异的单克隆抗体筛选[J].细胞与分子免疫学杂志,1998,14(3):212-216.doi:10.13423/j.cnki.cjcmi.002191.Hu M R,Sun Y X,Dong J X,Yu M,Shu C L.Indirect ELISA for cell-antigen-specific monoclonal antibody screening[J].J Cell Mol Immunol,1998,14(3):212-216.
[19] 王刚,张鹤晓,甘孟侯,张玉忠,王彩兰,崔向东,苏世敏,张兆平,姜永庄.IPMA检测猪生殖和呼吸综合征病毒抗体的研究[J].中国兽医杂志,1996,22(12):3-5.Wang G,Zhang H X,Gan M H,Zhang Y Z,Wang C L,Cui X D,Su S M,Zhang Z P,Jiang Y Z.Studies on the detection of antibodies to Porcine reproductive and respiratory syndrome virus using immunoperoxidase monolayer assay[J].China Veterinary Medicine,1996,22(12):3-5.
[20] Drew T W.Comparative serology of porcine reproductive and respiratory syndrome in eight European laboratories,using immunoperoxidase monolayer assay and enzyme-linked immunosorbent assay[J].Rev Sci Tech,1995,14(3):761-775.
[21] Deregt D,Prins S.A monoclonal antibody-based immunoperoxidase monolayer(micro-isolation)assay for detection of type 1 and type 2 bovine viral diarrhea viruses[J].Can J Vet Res,1998,62(2):152-155.
[22] Ooms K,Van G H,Botti S,Van Gaever T,Delputte P L,Nauwynck H J.Evaluation of viral peptide targeting to porcine sialoadhesin using a Porcine reproductive and respiratory syndrome virus vaccination-challenge model[J].Virus Res,2013,177(2):147-155.doi:10.1016/j.virusres.2013.07.019.
[23] Trus I,Bonckaert C,Vander M K,Nauwynck H J.Efficacy of an attenuated European subtype 1 Porcine reproductive and respiratory syndrome virus(PRRSV)vaccine in pigs upon challenge with the East European subtype 3 PRRSV strain Lena[J].Vaccine,2014,32(25):2995-3003.doi:10.1016/j.vaccine.2014.03.077.
[24] Huang L P,Lu Y H,Wei Y W,Guo L J,Liu C M.Identification of one critical amino acid that determines a conformational neutralizing epitope in the capsid protein of porcine circovirus type2[J].BMC Microbiol,2011,11:188.
[25] 唐娜,王金良,张春玲,董林,祖立闯,曲光刚,谢金文,张松林,柳增善,沈志强.猪繁殖与呼吸综合征病毒单克隆抗体的制备及鉴定[J].动物医学进展,2013,34(11):27-31.doi:10.16437/j.cnki.1007-5038.2013.11.018.Tang N,Wang J L,Zhang C L,Dong L,Zu L C,Qu G G,Xie J W,Zhang S L,Liu Z S,Shen Z Q.Development and identification of monoclonal antibodies against Porcine reproductive and respiratory syndrome virus[J].Progress in Veterinary Medicine,2013,34(11):27-31.
[26] Pileri E,Cortey M,Rodriguez F,Sibila M,Fraile L,Segalés J.Comparison of the immunoperoxidase monolayer assay and three commercial ELISAs for detection of antibodies against porcine circovirus type2[J].Vet J,2014,201(3):429-432.doi:10.1016/j.tvjl.2014.05.025.
[27] 李青梅,王丽,冯丽丽,张雨杭,赵东,杨艳艳,邢广旭,柴书军,李赛赛,张丽萍,郭军庆,张改平.鸡新城疫病毒中和单克隆抗体的制备及鉴定[J].河南农业科学,2017,46(1):127-131.doi:10.15933/j.cnki.1004-3268.2017.01.023.Li Q M,Wang L,Feng L L,Zhang Y H,Zhao D,Yang Y Y,Xing G X,Chai S J,Li S S,Zhang L P,Gui J Q,Zhang G P.Production and characterization of neutralizing monoclonal antibodies against Newcastle disease virus[J].Journal of Henan Agricultural Sciences,2017,46(1):127-131.
[28] Wensvoort G,Terpstra C,Boonstra J,Bloemraad M,Van Z D.Production of monoclonal antibodies against Swine fever virus and their use in laboratory diagnosis[J].Vet Microbiol,1986,12(2):101-108.
[29] 徐璇,董静,白娟,王先炜,姜平.两株抗猪伪狂犬病毒的单克隆抗体制备与鉴定[J].畜牧与兽医,2017,49(5):118-120.Xu X,Dong J,Bai J,Wang X W,Jiang P.Preparation and characterization of two monoclonal antibodies against Pseudorabies virus[J].Animal Husbandry & Veterinary Medicine,2017,49(5):118-120.
[30] Yoon K J,Zimmerman J J,Swenson S L,Mcginley M J,Eernisse K A,Brevik A,Rhinehart L L,Frey M L,Hill H T,Platt K B.Characterization of the humoral immune response to Porcine reproductive and respiratory syndrome(PRRS)virus infection[J].J Vet Diagn Invest,1995,7(3):305-312.
[31] Liang H,Wang H,Zhang L,Gu H,Zhang G.Development of a novel immunoperoxidase monolayer assay for detection of Swine Hepatitis E virus antibodies based on stable cell lines expressing the ORF3 protein[J].Acta Vet Hung,2014,62(2):243-256.doi:10.1556/AVet.2013.051.
[2] Collins J E,Benfield D A,Christianson W T,Harris L,Hennings J C,Shaw D P,Goyal S M,McCullough S,Morrison R B,Joo H S,et al.Isolation of Swine infertility and respiratory syndrome virus(isolate ATCC VR-2332)in North America and experimental reproduction of the disease in gnotobiotic pigs[J].J Vet Diagn Invest,1992,4(2):117.doi:10.1177/104063879200400201.
[3] Stevenson G W,Alstine W G V,Kanitz C L.Characterization of infection with endemic Porcine reproductive and respiratory syndrome virus in a swine herd[J].Journal of the American Veterinary Medical Association,1994,204(12):1938-1942.
[4] 郭敏,郎广平,冯娇,李彬,付云云,单悦,赵建增,高英杰.猪繁殖与呼吸综合征的研究进展[J].中国畜牧兽医,2013,40(4):211-215.Guo M,Lang G P,Feng J,Li B,Fu Y Y,Shan Y,Zhao J Z,Gao Y J.Research progress in porcine reproductive and respiratory syndrome[J].China Animal Husbandry and Veterinary Medicine,2013,40(4):211-215.
[5] Benfield D A,Nelson E,Collins J E,Harris L,Goyal S M,Robison D,Christianson W T,Morrison R B,Gorcyca D,Chladek D.Characterization of Swine infertility andrespiratory syndrome(SIRS)virus(isolate ATCC VR-2332)[J].J Vet Diagn Invest,1992,4(2):127-133.doi:10.1177/104063879200400202.
[6] 郭宝清,陈章水,刘文兴,崔益沫.从疑似PRRS流产胎儿分离 PRRSV的研究[J].中国畜禽传染病,1996,2:1-4.Guo B Q,Chen Z S,Liu W X,Cui Y M.Separation of PRRSV from fetus with suspected PRRS abortion[J].Chinese Livestock and Infectious Diseases,1996,2:1-4.
[7] 郭振华,陈鑫鑫,李睿,乔松林,郭军庆,张改平.我国猪繁殖与呼吸综合征病毒流行历史及现状[J].畜牧兽医学报,2018,49(1):1-9.doi:10.11843/j.issn.0366-6964.2018.01.001.Guo Z H,Chen X X,Li R,Qiao S L,Guo J Q,Zhang G P.The prevalence history and current status of porcine reproductive and respirotory syndrome in China[J].Acta Veterinaria et Zootechnica Sinica,2018,49(1):1-9.
[8] 代军,雷蕾,任志华.猪繁殖与呼吸综合征防控研究进展[J].动物医学进展,2014,35(4):97-101.doi:10.16437/j.cnki.1007-5038.2014.04.022.Dai J,Lei L,Ren Z H.Advances in research on prevention and control of porcine reproductive and respiratory syndrome[J].Progress in Veterinary Medicine,2014,35(4):97-101.
[9] Chaikhumwang P,Tantituvanont A,Tripipat T,Tipsombatboon P,Piriyapongsa J,Nilubol D .Dynamics and evolution of highly Porcine reproductive and respiratory syndrome virus following its introduction into a herd concurrently infected with both types 1 and 2[J].Infect Genet Evol,2015,30:164-174.doi:10.1016/j.meegid.2014.12.025.
[10] 杨汉春,周磊.猪繁殖与呼吸综合征病毒的遗传变异与演化[J].生命科学,2016,28(3):325-336.doi:10.13376/j.cbls/2016041.Yang H C,Zhou L.Genetic variation and evolution of Porcine reproductive and respiratory syndrome virus[J].Chinese Bulletin of Life Sciences,2016,28(3):325-336.
[11] Yang L,Yoon K J,LI Y,Lee J H,Zimmerman J J,Frey M L,Harmon K M,Platt K B .Antigenic and genetic variations of the 15 kD nucleocapsid protein of Porcine reproductive and respiratory syndrome virus isolates[J].Arch Virol,1999,144:525-546.
[12] Meulenberg J J,Petersen-Den Besten A,De Kiuyer E P,Moormann R J,Schaaper W M,Wensvoort G .Characterization of proteins encoded by ORFs 2 to 7 of Lelystad virus[J].Virology,1995,206:155-163.
[13] Wu W H,Fang Y,Farwell R,Christopher-Hennings J,Nelson E A.A 10-kDa structural protein of Porcine reproductive and respiratory syndrome virus encoded by ORF2b[J].Virology,2001,287:183-191.doi:10.1006/viro.2001.1034.
[14] Dea S,Gagnon C A,Mardassi H,Pirzadeh B,Rogan D.Current knowledge on the structural proteins of porcine reproductive and respiratory syndrome(PRRS)virus:comparison of the North American and European Isolates[J].Arch Virol,2000,145(4):659-688.
[15] Guo Z,Chen X X,Li R,Qiao S,Zhang G .The prevalent status and genetic diversity of Porcine reproductive and respiratory syndrome virus in China:A molecular epidemiological perspective[J].Virol J,2018,15:2.doi:10.1186/s12985-017-0910-6.
[16] Wang F X,Yang Y,Liu X,He M H,Liu Y,Sun N,Zhu H W,Ren J Q,Wu H,Wen Y J.Development of a monoclonal antibody for differentiating Porcine reproductive and respiratory syndrome virus and identification of a novel non-structural protein 2 epitope peptide[J].Virus Disease,2017,28(4):408-415.doi:10.1007/s13337-017-0400-x.
[17] Plagemann P G.Epitope specificity of monoclonal antibodies to the N-protein of Porcine reproductive and respiratory syndrome virus determined by ELISA with synthetic peptides[J].Vet Immunol Immunopathol,2005,104(1/2):59-68.doi:10.1016/j.vetimm.2004.10.004.
[18] 胡美茹,孙英勋,董家新,于鸣,舒翠玲.间接ELISA法用于细胞抗原特异的单克隆抗体筛选[J].细胞与分子免疫学杂志,1998,14(3):212-216.doi:10.13423/j.cnki.cjcmi.002191.Hu M R,Sun Y X,Dong J X,Yu M,Shu C L.Indirect ELISA for cell-antigen-specific monoclonal antibody screening[J].J Cell Mol Immunol,1998,14(3):212-216.
[19] 王刚,张鹤晓,甘孟侯,张玉忠,王彩兰,崔向东,苏世敏,张兆平,姜永庄.IPMA检测猪生殖和呼吸综合征病毒抗体的研究[J].中国兽医杂志,1996,22(12):3-5.Wang G,Zhang H X,Gan M H,Zhang Y Z,Wang C L,Cui X D,Su S M,Zhang Z P,Jiang Y Z.Studies on the detection of antibodies to Porcine reproductive and respiratory syndrome virus using immunoperoxidase monolayer assay[J].China Veterinary Medicine,1996,22(12):3-5.
[20] Drew T W.Comparative serology of porcine reproductive and respiratory syndrome in eight European laboratories,using immunoperoxidase monolayer assay and enzyme-linked immunosorbent assay[J].Rev Sci Tech,1995,14(3):761-775.
[21] Deregt D,Prins S.A monoclonal antibody-based immunoperoxidase monolayer(micro-isolation)assay for detection of type 1 and type 2 bovine viral diarrhea viruses[J].Can J Vet Res,1998,62(2):152-155.
[22] Ooms K,Van G H,Botti S,Van Gaever T,Delputte P L,Nauwynck H J.Evaluation of viral peptide targeting to porcine sialoadhesin using a Porcine reproductive and respiratory syndrome virus vaccination-challenge model[J].Virus Res,2013,177(2):147-155.doi:10.1016/j.virusres.2013.07.019.
[23] Trus I,Bonckaert C,Vander M K,Nauwynck H J.Efficacy of an attenuated European subtype 1 Porcine reproductive and respiratory syndrome virus(PRRSV)vaccine in pigs upon challenge with the East European subtype 3 PRRSV strain Lena[J].Vaccine,2014,32(25):2995-3003.doi:10.1016/j.vaccine.2014.03.077.
[24] Huang L P,Lu Y H,Wei Y W,Guo L J,Liu C M.Identification of one critical amino acid that determines a conformational neutralizing epitope in the capsid protein of porcine circovirus type2[J].BMC Microbiol,2011,11:188.
[25] 唐娜,王金良,张春玲,董林,祖立闯,曲光刚,谢金文,张松林,柳增善,沈志强.猪繁殖与呼吸综合征病毒单克隆抗体的制备及鉴定[J].动物医学进展,2013,34(11):27-31.doi:10.16437/j.cnki.1007-5038.2013.11.018.Tang N,Wang J L,Zhang C L,Dong L,Zu L C,Qu G G,Xie J W,Zhang S L,Liu Z S,Shen Z Q.Development and identification of monoclonal antibodies against Porcine reproductive and respiratory syndrome virus[J].Progress in Veterinary Medicine,2013,34(11):27-31.
[26] Pileri E,Cortey M,Rodriguez F,Sibila M,Fraile L,Segalés J.Comparison of the immunoperoxidase monolayer assay and three commercial ELISAs for detection of antibodies against porcine circovirus type2[J].Vet J,2014,201(3):429-432.doi:10.1016/j.tvjl.2014.05.025.
[27] 李青梅,王丽,冯丽丽,张雨杭,赵东,杨艳艳,邢广旭,柴书军,李赛赛,张丽萍,郭军庆,张改平.鸡新城疫病毒中和单克隆抗体的制备及鉴定[J].河南农业科学,2017,46(1):127-131.doi:10.15933/j.cnki.1004-3268.2017.01.023.Li Q M,Wang L,Feng L L,Zhang Y H,Zhao D,Yang Y Y,Xing G X,Chai S J,Li S S,Zhang L P,Gui J Q,Zhang G P.Production and characterization of neutralizing monoclonal antibodies against Newcastle disease virus[J].Journal of Henan Agricultural Sciences,2017,46(1):127-131.
[28] Wensvoort G,Terpstra C,Boonstra J,Bloemraad M,Van Z D.Production of monoclonal antibodies against Swine fever virus and their use in laboratory diagnosis[J].Vet Microbiol,1986,12(2):101-108.
[29] 徐璇,董静,白娟,王先炜,姜平.两株抗猪伪狂犬病毒的单克隆抗体制备与鉴定[J].畜牧与兽医,2017,49(5):118-120.Xu X,Dong J,Bai J,Wang X W,Jiang P.Preparation and characterization of two monoclonal antibodies against Pseudorabies virus[J].Animal Husbandry & Veterinary Medicine,2017,49(5):118-120.
[30] Yoon K J,Zimmerman J J,Swenson S L,Mcginley M J,Eernisse K A,Brevik A,Rhinehart L L,Frey M L,Hill H T,Platt K B.Characterization of the humoral immune response to Porcine reproductive and respiratory syndrome(PRRS)virus infection[J].J Vet Diagn Invest,1995,7(3):305-312.
[31] Liang H,Wang H,Zhang L,Gu H,Zhang G.Development of a novel immunoperoxidase monolayer assay for detection of Swine Hepatitis E virus antibodies based on stable cell lines expressing the ORF3 protein[J].Acta Vet Hung,2014,62(2):243-256.doi:10.1556/AVet.2013.051.