Preparation and identification of monoclonal antibody against Zaire Ebola virus
LIU Rong-jiao1, WANG Ren-xia3, LI Zi-wei1, BIN Lei1, TAN Ya-fang2, WANG Jin2, DU Zong-min2, DENG Zhong-liang1
1. School of Public Health, University of South China, Hengyang 421001, China; 2. Laboratory of Analytical Microbiology, State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military MedicalSciences, Beijing 100071, China; 3. Clinical Laboratory of Center for Disease Prevention and Control of Zhuzhou,Zhuzhou 412000, China
Abstract:Zaire Ebola virus nucleoprotein (EBOV NP) synthetic peptide was used as the immunogen to prepare mouse monoclonal antibody, and the specificity of prepared monoclonal antibody was tested with recombinant nucleoproteins of four Ebola virus epidemic strains.The artificially synthesized nucleoprotein peptides of Zaire Ebola virus were used to immunize animals, and hybridoma cell lines were screened after the cells fusion.Four eukaryotic expression vectors of the Ebola virus subtype were constructed to transfect HEK293T cell to express target proteins. Then the eukaryotic expression proteins were used as antigens to test the specificity of prepared monoclonal antibody against Zaire EBOV by Western blotting. We accomplished the preparation of monoclonal antibody against Zaire EBOV, and twelve cell lines that can secret monoclonal antibodies against Zaire EBOV were obtained,the titers of antibodies in mouse ascites were among 1∶104-1∶105. Three hybridoma cell lines of the twelve were screened, which secret antibodies that have no cross reaction with other three Ebola virus subtype, and these antibodies had high titer and specificity. In conclusion, the monoclonal antibody against Zaire Ebola virus was successfully prepared, which laid the foundation for the rapid detection of Ebola virus.
刘荣娇, 王仁霞, 李子微, 宾蕾, 谭亚芳, 王津, 杜宗敏, 邓仲良. 抗扎伊尔型埃博拉病毒单克隆抗体的制备和鉴定[J]. 中国人兽共患病学报, 2018, 34(7): 619-623.
LIU Rong-jiao, WANG Ren-xia, LI Zi-wei, BIN Lei, TAN Ya-fang, WANG Jin, DU Zong-min, DENG Zhong-liang. Preparation and identification of monoclonal antibody against Zaire Ebola virus. Chinese Journal of Zoonoses, 2018, 34(7): 619-623.
[1] Vuren PJ, Grobbelaar A, Storm N, et al.Comparative evaluation of the diagnostic performance of the prototype cepheid GeneXpert Ebola assay[J]. J Clin Microbiol,2016, 54(2): 359-367. DOI: 10.1128/JCM.02724-15 [2] Baize S, Pannetier D, Oestereich L, et al.Emergence of Zaire Ebola virus disease in Guinea[J]. New N Engl J Med, 2014, 371(15): 1418-1425. DOI: 10.1056/NEJMoa1404505 [3] Wan W, Kolesnikova L, Clarke M, et al.Structure and assembly of the Ebola virus nucleocapsid[J]. Nature, 2017, 551(7680): 394-397. DOI: 10.1038/nature24490 [4] Takada A, Robison C, Goto H, et al.A system for functional analysis of Ebola virus glycoprotein[J]. Proc Natl Acad Sci U S A, 1997, 94(26): 14764-14769. DOI: 10.1073/pnas.94.26.14764 [5] Baker L, Ellena J, Handing K, et al.Molecular architecture of the nucleoprotein C-terminal domain from the Ebola and Marburg viruses[J]. Acta Crystallogr D Struct Biol, 2016, 72(Pt 1): 49-58. DOI: 10.1107/S2059798315021439 [6] Feldmann H, Klenk HD, Sanchez A.Molecular biology and evolution of filoviruses[J]. Arch virol suppl, 1993, 7(7): 81. DOI: 10.1007/978-3-7091-9300-6_8 [7] Henderson WW, Monroe MC, St Jeor SC, et al.Naturally occurring Sin Nombre virus genetic reassortants[J]. Virology, 1995, 214(2): 602-610. DOI: 10.1006/viro.1995.0071 [8] Feldmann H, Kiley MP.Classification, structure, and replication of filoviruses[J].Curr Top Microbiol Immunol, 1999, 235(235): 1-21. DOI: 10.1007/978-3-642-59949-1_1 [9] Tao W, Gan T, Guo M, et al.Novel stable Ebola virus minigenome replicon reveals remarkable stability of the viral genome[J]. J Virol, 2017, 91(22). DOI: 10.1128/JVI.01316-17 [10] Feldmann H, Geisbert TW.Ebola hemorrhagic fever[J]. Lancet, 2010, 377(9768): 849-862. DOI: 10.1016/S0140-6736(10)60667-8 [11] Saijo M, Niikura M, Morikawa S, et al.Enzyme-linked immunosorbent assays for detection of antibodies to Ebola and Marburg viruses using recombinant nucleoproteins[J]. J Clin Microbiol, 2001, 39(1): 1-7. DOI: 10.1128/JCM.39.1.1-7.2001 [12] Sochas L, Channon A, Nam S. Counting indirect crisis-related deaths in the context of a low-resilience health system: the case of maternal and neonatal health during the Ebola epidemic in Sierra Leone[J]. Health Policy Plan, 2017, 32(suppl3): iii32-iii39. DOI: 10.1093/heapol/czx108 [13] Russo C, Callegaro L, Lanza E, et al.Re.: Purification of IgG monoclonal antibody by caprylic acid precipitation[J]. J Immunol Methods, 1983, 65(1/2): 269. DOI: 10.1016/0022-1759(83)90324-1 [14] Mckinney M, Parkinson A.A simple, non-chromatographic procedure to purify immunoglobulins from serum and ascites fluid[J]. J Immunol Methods, 1987, 96(2): 271-278. DOI: 10.1016/0022-1759(87)90324-3 [15] Ksiazek TG, Rollin PE, Williams AJ, et al.Clinical virology of Ebola hemorrhagic fever (EHF): virus, virus antigen and IgG and IgM[J]. J Infect Dis, 1999, 179(2): S177. DOI: 10.1086/514321 [16] 梁云浩. 非洲猪瘟病毒蛋白p54原核真核表达、间接ELISA检测方法建立及单克隆抗体制备[D]. 广州:暨南大学, 2014. [17] 汪孟航, 朱洪伟, 刘莹, 等. 狂犬病病毒G蛋白V区特异性单克隆抗体的制备与鉴定[J]. 中国兽医科学, 2017,57(5): 592-596.