鸭肠炎病毒单克隆抗体的制备

Preparation of monoclonal antibodies against duck enteritis virus

采用差速离心和蔗糖密度梯度离心法提纯鸭肠炎病毒 (DEV)鸭胚成纤维细胞适应毒 ,免疫BALB/c小鼠 ,取脾细胞与SP2 / 0骨髓瘤细胞融合 ,经间接ELISA筛选 ,有限稀释法 3次克隆 ,获得了 2株稳定分泌DEV单克隆抗体的杂交瘤细胞株 1B6和 2G8。经鉴定 ,2株单克隆抗体分别为IgM和IgG1 亚类 ,腹水ELISA效价均达到 1∶1 0 7以上。以 2G8单抗腹水为材料 ,采用间接免疫荧光抗体技术对DEV标准毒感染的鸭胚成纤维细胞进行检测 ,结果表明 ,用该抗体可特异性检出接毒后 6h感染细胞中的DEV ,且感染后 4 8h免疫荧光强度最高 ,该单抗与细胞成分和鸭肝炎病毒无交叉反应。将杂交瘤细胞冻存 3和 6个月 。

Duck enteritis virus(DEV)was propagated in duck embryo fibroblasts(DEF) and purified by ultracentrifugation. Four BALB/c mice were immunized subcutaneously with purifiedDEV antigen three times at 10 days interval. The immunized spleen cellsof the mice were fused with the SP2/0 myeloma cells. An indirect ELISA was developed to detect DEV antigen-specific monoclonal antibodies(McAb) in cell culture supernatants. Two hybridoma cell lines, named 1B_(6)and 2G_(8 ),secreting McAb against DEV were prepared after three times of limiting-dilution cloning. The immunoglobulin subclass of 1B_(6)and 2G_(8 )was IgM and IgG1 respectively. The ELISA titer of ascites fluid collected from mice inoculated with either of the two hyridomas were all above 1∶10^(7). A McAb based indirect immunoflurescent antibody assay was established to detect DEV antigen in DEF using purified 2G_(8)ascites immunoglobulin. The optimal working dilution of the McAb was 1∶100. The viral antigen could be detected out in 6 hours post-inoculation. The immunofluorescence was most significant with relativelyintact cell morphorology.