单抗介导的斑点免疫金渗滤法（DIGFA）快速检测禽流感病毒
摘要
将禽流感病毒(AIV H_5N_1、H_9N_2)纯化制成抗原，应用淋巴细胞杂交瘤技术，通过ELISA筛选，建立了1株抗禽流感病毒的杂交瘤细胞株：2C_8，其分泌抗体能力稳定，经Western-blotting鉴定为针对NP蛋白的单抗，免疫球蛋白亚类属于IgG_1。分泌的单克隆抗体与AIV H_5N_1、H_9N_2各毒株和A型SIV H_1N_1、H_3N_2均发生反应，但对鸡NDV、IBDV、EDSV及IBV均无交叉反应，表明该单抗具A型流感群特异性。此杂交瘤细胞上清、腹水ELISA效价分别为2~7、2~(11)，但无血凝抑制性和中和活性，对热稳定性良好。腹水经辛酸-硫酸铵沉淀法纯化后，蛋白含量为2.113mg／ml。此单克隆抗体的获得为A型流感病毒病原的研究、流感快速诊断方法的建立奠定了基础。
利用获得的单克隆抗体和胶体金标记技术，在国内首先建立了快速检测AIV的DIGFA(夹心法)。检测时先将单抗点样于NCM中央，封闭后与待检抗原作用10min，洗涤后再与胶体金标记纯化的兔抗AIVIgG作用5min，洗涤后观察。在膜上出现红色斑点的为阳性，不出现斑点为阴性。用此法检测AIV H_5N_1、H_9N_2、SIV毒株均为阳性，而NDV、IBDV、EDSV及IBV等均为阴性；对AIV H_9N_2纯化抗原及含毒尿囊液最低检出量分别为1.799ng／点(2μl)、10~3EID_(50)；检测AIV H_9N_2在鸡胚中繁殖时以66-92h含毒量最高；检测H_9N_2 SPF攻毒鸡泄殖腔棉拭子，至少感染后2-8天内可检出病毒，与病毒分离结果一致；对H_5N_1攻毒死亡鸡气管的病毒检出率80％；自然感染鸡气管10份能检出9份，与病毒分离的阳性符合率在90％以上。实践证明该法具有A型流感群特异性，可用于AIV早期诊断，其快速、敏感、简便，不需特殊设备、30分钟就能判定结果的特点，更适于基层实验室或现场应用。
AIV H9N2 or H5N1 was grown in chicken embryonated eggs and purified using 25000rmp centrifuge. Eight-week Balb/c mice were immunized according to Kazuhiro's procedure.A hybridoma named 2Cg was generated by fusion of NSo myelomas and Splenocytes from immunized mice . The monoclonal antibody( McAb) produced was screened by ELISA and proved to be IgG1 by AGP, Mouse ascitic fluid(ELISA titre:211) was purified (protein concentration:2.113mg/ml) and proved to react with NP protein in western-blotting. The result of cross-reaction in ELISA demonstrated that the McAb was group-specific.
The purified rabbit anti-AIV IgG was labeled with colloidal gold particals of lOnm in diameter . A McAb-mediated DIGFA was developed for detection of AIV. When AIV was to be detected, McAb anti-NP (1:320 diluted) was used to coat nitrocellulose membrane(NCM) and react with AIV for 20 minutes.After being washed,the NCM was immersed in the solution of rabbit anti- AIV IgG(l:64 diluted) for 5 minutes. The sample with obvious red dot was judged as positive reaction for AIV,otherwise,negative result.The detection limit of purified AIV antigen and allantioic fluid of AIV were 1.799ng each dot and 103EIDso respectively.AIV titre in inoculated eggs was highest from 66 to 92 hours postinoculation(p.i.).AIV could be 100% detected in cloacal swabs of specific-pathogen-free(SPF) challenged chickens 2 DPI and 80%- 90% detected in trachea of naturally infected or experimentally challenged chickens by DIGFA.We conclude that DIGFA was a rapid ^ sensitive N simple and non-labor-intensive test for
61
detecting type-A influenza virus in clinical specimens and could be applied to antigen surveillance of avian influenza especially in clinical diagnosis of primary laboratory.
利用获得的单克隆抗体和胶体金标记技术，在国内首先建立了快速检测AIV的DIGFA(夹心法)。检测时先将单抗点样于NCM中央，封闭后与待检抗原作用10min，洗涤后再与胶体金标记纯化的兔抗AIVIgG作用5min，洗涤后观察。在膜上出现红色斑点的为阳性，不出现斑点为阴性。用此法检测AIV H_5N_1、H_9N_2、SIV毒株均为阳性，而NDV、IBDV、EDSV及IBV等均为阴性；对AIV H_9N_2纯化抗原及含毒尿囊液最低检出量分别为1.799ng／点(2μl)、10~3EID_(50)；检测AIV H_9N_2在鸡胚中繁殖时以66-92h含毒量最高；检测H_9N_2 SPF攻毒鸡泄殖腔棉拭子，至少感染后2-8天内可检出病毒，与病毒分离结果一致；对H_5N_1攻毒死亡鸡气管的病毒检出率80％；自然感染鸡气管10份能检出9份，与病毒分离的阳性符合率在90％以上。实践证明该法具有A型流感群特异性，可用于AIV早期诊断，其快速、敏感、简便，不需特殊设备、30分钟就能判定结果的特点，更适于基层实验室或现场应用。
AIV H9N2 or H5N1 was grown in chicken embryonated eggs and purified using 25000rmp centrifuge. Eight-week Balb/c mice were immunized according to Kazuhiro's procedure.A hybridoma named 2Cg was generated by fusion of NSo myelomas and Splenocytes from immunized mice . The monoclonal antibody( McAb) produced was screened by ELISA and proved to be IgG1 by AGP, Mouse ascitic fluid(ELISA titre:211) was purified (protein concentration:2.113mg/ml) and proved to react with NP protein in western-blotting. The result of cross-reaction in ELISA demonstrated that the McAb was group-specific.
The purified rabbit anti-AIV IgG was labeled with colloidal gold particals of lOnm in diameter . A McAb-mediated DIGFA was developed for detection of AIV. When AIV was to be detected, McAb anti-NP (1:320 diluted) was used to coat nitrocellulose membrane(NCM) and react with AIV for 20 minutes.After being washed,the NCM was immersed in the solution of rabbit anti- AIV IgG(l:64 diluted) for 5 minutes. The sample with obvious red dot was judged as positive reaction for AIV,otherwise,negative result.The detection limit of purified AIV antigen and allantioic fluid of AIV were 1.799ng each dot and 103EIDso respectively.AIV titre in inoculated eggs was highest from 66 to 92 hours postinoculation(p.i.).AIV could be 100% detected in cloacal swabs of specific-pathogen-free(SPF) challenged chickens 2 DPI and 80%- 90% detected in trachea of naturally infected or experimentally challenged chickens by DIGFA.We conclude that DIGFA was a rapid ^ sensitive N simple and non-labor-intensive test for
61
detecting type-A influenza virus in clinical specimens and could be applied to antigen surveillance of avian influenza especially in clinical diagnosis of primary laboratory.
引文
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3．中国农业百科全书．兽医卷．农业出版社．1993
4. Schafer W. Vergleichende sero-immunologische unter suchunges uber die viren der influenza and klassichen Gefluegelpest Z Nature forsch 1955,10b:81-91
5. Lang. G A Isolation.of an unidentified hemagglutinating virus from the respiratory tract of turkeys. Avian Dis(9) 1965:495-504
6. Halvorson D A. Epizootiology of avian influenza-simultaneous monitoring of sentinel ducks and turkeys in Minnesota. Avian Ois1983(27):77-85
7．卡尔尼克．BW主编．禽病学．第九版[M]．高福、刘文军主译，北京：农业出版社，1999，746．
8. Guo Y J, et al. Characterization of a new avian-like influenza A virus from horses in China. Virology, 1992, 188:245
9. Reid AH, Fanning TG, Hultin JV, etal. Origin and evolution of the 1918 "Spanish" influenza virus hemagglutinin gene[J].Proc Natl Acad Sci USA, 1999,96(4):1651.
10. Capua I, Alexander DJ. Avian influenza and human health. Acta Trop 2002 Jul;83(1):1-6
11. Horimoto T Pandemic threat posed by avian influenza A viruses. Clin Microbiol Rev 2001 Jan;14(1):129-49
12. Suarez DL Sequence analysis of related ]ow-pathogenic and highly pathogenic H5N2 avian influenza isolates from United States live bird markets and poultry farms from 1983 to 1989. Avian Dis 2000 Apr-Jun;44(2):356-64
13. Karunakaran D Influenza A outbreaks in Minnesota turkeys due to subtype H10N7 and possible transmission by waterfowl. Avian Dis 1983 Apr-Jun;27(2):357-66
14．杨俊兴等．高致病性鸭流感病毒的分离与鉴定．中国畜牧兽医学会禽病学分会第十一次学术探讨会论文集．2002；92-93
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