【作者】 姜永忠；

【导师】 王明丽； 龚蕴贞； 史百芬；

【作者基本信息】 安徽医科大学 ， 病原生物学， 2002， 硕士

【摘要】 目的 制备具有中和人巨细胞病毒(HCMV)感染作用的单克隆抗体并对其进行生物学活性鉴定。探讨单克隆抗体在临床上的诊断和治疗作用。方法 用HCMVAD169株抗原免疫Balb／c小鼠5-6次后，取致敏的脾细胞和HGPRT SP2／0小鼠骨髓瘤细胞进行融合，在HAT选择培养基中培养二周，间接ELISA法筛选阳性克隆，挑取分泌抗体的杂交瘤株：再用间接ELISA进行鉴定，最终获得抗HCMV的阳性杂交瘤细胞4株，用琼脂糖免疫双扩散法鉴定McAbs的亚类。取腹水经盐析法初次提纯HCMV McAbs。Lowry法测定蛋白质浓度。在第6代HF细胞株上进行微量中和试验；同时用辣根过氧化物酶(HRP)标记D7株McAb。结果 获得了4株杂交瘤细胞株，制备的McAbs经间接ELISA和琼脂双扩散试验证实均能和HCMV抗原结合，显示高度特异性。McAbs经盐析后D7株蛋白质浓度为11． 2mg／ml，B5株蛋白质浓度13． 8mg／ml。其中D7株杂交瘤分泌的McAb经间接ELISA和Western-blotting试验证明能和HCMV pp65抗原特异性结合。微量中和实验亦证实该McAb具有中和抗体的性质。经HRP标记的该McAb(中和效价≥1：128) 还能与细胞培养中的AD169株HCMV发生特异性核内及胞浆内染色反应，且比间接免疫酶染法更快速、敏感、简便。结论 通过杂交瘤技术制备的抗HCMVpp65 McAb能和HCMV特异性结合并能在体外中和HCMV致细胞病变效应，为临床快速、准确诊断HCMV活动性感染提供了有力试剂；抗为McAb作为HCMV治疗药物的研发奠定了基础。更多还原

【Abstract】 Objective We produced neutralizing monoclonal antibody against HCMV infection and identified its biological character.We explored McAb effection in clinical diagnoses and therapy. Methods Balb/c mice were immunized by antigen of HCMV for five or six times. HGPRT" SP2/0 was fused with mice spleen B lymphocytes. The hybridoma cell was cultured in the HAT selective medium .The positive clone was screened by indirect ELISA(IELISA). We gathered supernatant of hybridoma and identified cell with IELISA .We finally got the four hybridoma strain which secreted antibody against HCMV. McAb class and subclass were identified by agarose immunity double diffusion technique. We inoculated the hybridoma into Intraperitoneal mice and hydroperitoneum was purified with salt precipitation. The protein concentration was detected by Lowry. We took micronutralization experiment in HE D7 hybridoma strain McAb was labeled by HRP. Results We produced monoclonal antibody against HCMV .The McAbs were demonstrated to combined HCMV antigen by IELISA and agarose double diffusion. Its possess high specificity. The protein concentration was 11.2mg/ml and 13.8mg/ml. The McAb which D7 hybridoma strain secrete was demonstrated specifically to bind HCMV pp65 antigen by IELISA and Western-blotting. Microneutralization also confirm the McAb character which has neutralizative antibody. The titer of McAb that was labeled by HRP was 1:128. Cone I us i ons HCMV pp65 monoclonal antibody which was produced by hybridoma technique can neutralize CPE of HCMV, which extended the way that monoclonal antibody were regard as drug.更多还原