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目的制备和鉴定鼠抗虾主要变应原TM的单克隆抗体(Monoclonal Antibody,McAb)。方法用重组TM蛋白为免疫原,免疫Balb/c小鼠,取免疫小鼠脾细胞与小鼠骨髓瘤NS-1细胞融合。间接ELISA法筛选特异性分泌的杂交瘤细胞。杂交瘤细胞株诱导小鼠产生腹水,再用蛋白A亲和层析法纯化抗体。采用Ig类与亚类鉴定试剂盒鉴定该单克隆抗体的Ig亚型;通过间接ELISA、Western Blotting鉴定该单克隆抗体的特性和交叉性,对常见食物的检测。结果获得4株可稳定分泌鼠抗虾主要变应原TM的单克隆抗体,其Ig亚型均为IgG1。且4株单抗均具有良好的效价。ELISA和Western Blotting分析表明该4株单抗均能识别重组TM蛋白和天然的虾提取物。结论成功制备了4株鼠抗虾主要变应原TM的单克隆抗体,为建立TM检测及纯化方法奠定了基础。
Objective To prepare and identify Monoclonal Antibody (McAb) against mouse anti-shrimp major allergen TM. Methods Balb / c mice were immunized with recombinant TM protein and the splenocytes of immunized mice were fused with mouse myeloma NS-1 cells. Indirect ELISA screening of specific secretion of hybridoma cells. The hybridoma cell line induces ascites in mice and the antibody is purified by protein A affinity chromatography. The Ig subtype of the monoclonal antibody was identified by Ig kit and subclass identification kit. The identity and cross-talk of the monoclonal antibody was identified by indirect ELISA and Western Blotting, and the common foods were detected. Results Four monoclonal antibodies that could stably secrete the major antigenic allergen TM of murine shrimp were obtained, and all of their Ig subtypes were IgG1. And four monoclonal antibodies have good potency. ELISA and Western Blotting analysis showed that all four monoclonal antibodies recognized recombinant TM protein and natural shrimp extract. Conclusion Four monoclonal antibodies against the major allergens TM of murine shrimp were successfully prepared, which laid the foundation for the establishment of the TM detection and purification methods.