人巨细胞病毒单克隆抗体的制备及其体外中和活性鉴定  

Preparation of monoclonal antibody against HCMV and identification of neutralizing activity in vitro

作　　者：姜永忠[1] 王明丽[1] 李艳秋[1] 余莉[1] 胡勇[1] 毕克菊[1] 

机构地区：[1]安徽医科大学微生物学教研室,合肥230032

出　　处：《安徽医科大学学报》2002年第3期169-172,共4页Acta Universitatis Medicinalis Anhui

基　　金：安徽省科学技术厅生物医学"十五"重大攻关项目 (编号0 13 0 3 0 0 3 )

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。方法　用HCMV抗原免疫Balb/c小鼠 5、6次后 ,取致敏的脾脏B淋巴细胞和HGPRT SP2 /0小鼠骨髓瘤细胞进行融合 ,在HAT选择培养基中培养 2周 ,间接ELISA法筛选克隆。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的阳性杂交瘤细胞株 ,用Ouchterlony法鉴定McAb的亚类。小鼠腹腔内接种杂交瘤细胞 ,取腹水经盐析法提纯IgG。Lowry法测定蛋白质浓度 ,在第 6代HF细胞株及原代培养新生乳鼠脑神经细胞上进行微量中和实验。结果　获得了 4株杂交瘤细胞株 ,制备的F9McAbs盐析后蛋白质浓度为 11 2g/L ,血清学试验证明该McAbs能和HCMV抗原进行特异性结合 ,制备的IgG具有高度特异性。且体外实验已证实 ,该McAb具有中和抗体的性质。结论　通过杂交瘤技术制备的HCMV单克隆抗体能在体外中和HCMV致病变效应 。Objective The neutralizing monoclonal antibody was prepared against HCMV infection and its bio logical character identified. Methods Balb/c mice were immunized by antigen of HCMV for 5 or 6 times. HGPRT SP2/0 is fused with mouse spleen B lymphocytes. The hybridoma cell is cultured in HAT selective medium. The clone was screened by indirect ELISA(IELISA), then selecting the hybridoma of secreting antibody. The supernatant of hybridoma was collected and cells were identified with IELISA. The hybridoma secreting antibody against HCMV was obtained. McAb class and subclass is identified by Ouchterlony technique. We inoculate intraperitoneally the hybridoma into mouse and purify seroperitoneum with salt precipitation. The protein concentration was detected by Lowry method. The nutralization experiment was done in the sixth generation HF and primary natal mice nerve. Results The monoclonal antibody against HCMV was produced and protein concentration is 11 2 mg/ml. McAb specifically combined HCMV antigen. Subclass of McAb is IgG1 and had high specificity. Conclusion HCMV monoclonal antibody produced by hybridoma technique can neutralize CPE of HCMV ,which expands the way that monoclonal antibody are regard as drugs.

关 键 词：人巨细胞病毒单克隆抗体 制备 体外中和活性 鉴定 

分 类 号：R392－33[医药卫生—免疫学]