基孔肯亚病毒中和单克隆抗体的筛选与鉴定  

Screening and identification of neutralizing monoclonal antibodies against Chikungunya virus

作　　者：步恒富[1] 吴庆丽 祝庆余[1] 蒋越 郝盈 杨保安[1] 江志奎[2] 李钟铎[1] 

机构地区：[1]军事医学科学院微生物学流行病学研究所,北京100850 [2]解放军第105医院,合肥230031

出　　处：《军事医学科学院院刊》2000年第1期16-18,共3页Bulletin of the Academy of Military Medical Sciences

基　　金：总后"九五"指令性课题资助!项目 (96L0 2 0 )

摘　　要：目的 :建立抗基孔肯亚病毒单克隆抗体细胞株 ,筛选具有中和活性的特异性单克隆抗体。方法 :利用细胞融合技术建立抗基孔肯亚病毒单克隆抗体杂交瘤细胞株 ,用细胞培养中和试验初筛具有中和活性的单克隆抗体 ,用固定单克隆抗体稀释病毒方法进行乳鼠中和试验 ,进一步验证单克隆抗体的保护作用 ;用中和交叉试验鉴定中和单克隆抗体反应的特异性。结果 :用免疫荧光法检测建立了 9株能稳定分泌抗基孔肯亚病毒单克隆抗体的杂交瘤细胞株 ,初筛出 4株具有中和活性的单克隆抗体 ,其中 1F1单克隆抗体稀释 2 0 0倍时可保护 50 %细胞不产生细胞病变 ;乳鼠中和试验1F1单克隆抗体中和指数为 10 3.3,对试验小鼠有较强的保护作用。中和交叉试验表明 ,1F1单克隆抗体只中和基孔肯亚病毒 ,与其他病毒无交叉反应 ,特异性较高。结论 :制备了 9株抗基孔肯亚病毒单克隆抗体杂交瘤细胞株 ,筛选得到了 1F1具有较强中和活性的特异性单克隆抗体 ,可望为基孔肯亚病毒病的诊断。Objective: To establish hybridomas secreting monoclonal antibodies (McAbs) against Chikungunya virus and to screen neutralizing McAbs against Chikungunya virus. Methods: The hybridomas were established by cell fusion and indirect immunofluorescence assay and used to identify and characterize the viral neutralizing ability by cell culture neutralizing test (CCNT). Sucking mouse neutralizing test (SmNT) was used to verify animal protection ability by fixing McAbs and diluting virus methods. Neutralizing crossing test (NCT) was used to identify the specificity of neutralizing McAbs in CCNT. Results: Nine hybridomas secreting McAbs against Chikungunya virus were established. In CCNT , 1F 1 McAb of 1∶200 dilution inhibited 50% cell pathogenic effect (CPE) in BHK 13 cells after mixing with equal volume of 100TCID 50 virus and incubation in 5% CO 2 incubator for 1h, 37℃. 1F 1 McAb presented high specificity for neutralizing Chikungunya virus only and showed no reaction with other virus in NCT. In SmNT, 1F 1 McAb neutralizing value reached 10 3.3 and prevented sucking mice from death. Conclusion: Nine hybridomas secreting McAbs against Chikungunya virus were established , and 1F 1 neutralizing McAb was with a very high specificity in CCNT obtained through screening.It is expected that the 1F 1 McAb will provide good preventive and curative agents for of Chikungunya infectious disease.

关 键 词：基孔肯亚病毒 单克隆抗体 筛选 鉴定 

分 类 号：R373[医药卫生—病原生物学]