【作者】 黄娟；

【导师】 王泽霖；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2003， 硕士

【摘要】 将禽流感病毒（AIV H₅N₁、H₉N₂）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：2C₈，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IgG₁。分泌的单克隆抗体与AIV H₅N₁、H₉N₂各毒株和A型SIV H₁N₁、H₃N₂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2⁷、2¹¹，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 H₅N₁、H₉N₂、SIV毒株均为阳性，而NDV、IBDV、EDSV及IBV等均为阴性；对AIV H₉N₂纯化抗原及含毒尿囊液最低检出量分别为1.799ng／点（2μl）、10³EID₅₀；检测AIV H₉N₂在鸡胚中繁殖时以66-92h含毒量最高；检测H₉N₂ 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H₅N₁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。更多还原

【Abstract】 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 for61detecting type-A influenza virus in clinical specimens and could be applied to antigen surveillance of avian influenza especially in clinical diagnosis of primary laboratory.更多还原