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采用1,1’-羰基二咪唑(CDI)活化法成功将脱氧雪腐镰刀菌烯醇分子上C-3和C-15位羟基与载体蛋白赖氨酸的氨基共价偶联得到免疫原DON-BSA和包被原DON-OVA,并用三硝基苯磺酸法对合成结果进行鉴定。以人工抗原免疫Balb/c小鼠,取小鼠脾细胞与SP2/O鼠骨髓瘤细胞融合,经筛选和多次亚克隆,得到了1株能稳定分泌脱氧雪腐镰刀菌烯醇抗体的单克隆细胞株1D5,并制备单克隆抗体腹水。经检测该抗体亚型为IgG1,亲和力常数Ka为8.33×107L/mol,交叉反应的试验结果显示该抗体与其他真菌毒素无交叉反应率,稳定性良好,为粮油食品中脱氧雪腐镰刀菌烯醇免疫快速检测技术建立及产品研发提供了关键试剂材料。
The covalent coupling of C-3 and C-15 hydroxyl groups on the deoxynivalenol to the amino group of the carrier protein lysine was successfully carried out by the 1,1’-carbonyldiimidazole (CDI) activation method to obtain the immunogen DON -BSA and coated DON-OVA, and the synthesis results were identified by trinitrobenzene sulfonic acid method. Balb / c mice were immunized with artificial antigen. The mouse spleen cells were fused with SP2 / O mouse myeloma cells. After screening and multiple subcloning, a monoclonal antibody that could stably secrete deoxynivalenol Cell line 1D5 was cloned and monoclonal antibody ascites was prepared. The antibody was subcloned into IgG1 and the affinity constant Ka was 8.33 × 107 L / mol. The cross-reactivity test showed that the antibody did not cross-react with other mycotoxins and had good stability. The results showed that the antibody was stable against deoxynivalenol Alcohol immunoassay rapid detection technology and product development to provide a key reagent material.