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目的制备和鉴定鼠抗平榛主要变应原Cor h 1的单克隆抗体(Monoclonal Antibody,McAb)。方法用重组Cor h 1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。通过间接ELISA法筛选分泌特异性McAb的杂交瘤细胞。用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McAbs的特性和交叉性。结果获得4株可稳定分泌鼠抗平榛主要变应原Cor h 1的McAbs,其Ig亚型均为IgG1,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Cor h 1蛋白,其中3株单抗能够识别天然平榛提取物。结论成功制备了4株鼠抗平榛主要变应原Cor h 1的单克隆抗体,为建立平榛主要变应原Cor h 1检测及纯化方法奠定了基础。
Objective To prepare and identify Monoclonal Antibody (McAb) against Corh 1, a major allergen of Cinnabarin. Methods Balb / c mice were immunized with the recombinant protein Cor h 1 and the splenocytes of immunized mice were fused with mouse myeloma NS-1 cells. Hybridoma cells secreting specific McAbs were screened by indirect ELISA. Mice were induced to produce ascites by hybridoma cell lines and antibodies were purified by protein A affinity chromatography. The Ig subtype of the monoclonal antibody was identified by Ig class and subclass identification kit. The McAbs were characterized by indirect ELISA and Western Blotting. RESULTS: Four McAbs that secreted Corh 1, a major allergen of C. hazelnse, were successfully secreted and their Ig subtypes were IgG1. All of them had good titer. ELISA and Western Blotting analysis showed that these McAbs were able to recognize Recombinant Cor h 1 protein, of which 3 monoclonal antibodies to identify natural Hazel extract. Conclusion Four monoclonal antibodies against Corh 1, the major allergen of Rhizoma Corydalis, were successfully prepared, which laid the foundation for the establishment of Corh 1 detection and purification method.