相思子毒素单克隆抗体的制备纯化及鉴定

目的制备相思子毒素单克隆抗体并鉴定其特性。方法以甲醛处理的相思子毒素毒蛋白为抗原免疫BALB/c小鼠;取免疫小鼠的脾细胞与Sp2/0骨髓瘤细胞融合,经间接ELISA法筛选、融合细胞有限稀释法克隆、克隆化杂交瘤细胞株的亚类鉴定等方法筛选出单克隆抗体杂交瘤细胞株,用杂交瘤细胞株诱生小鼠腹水,应用蛋白A亲和层析法进行单抗的纯化,并对单克隆抗体的特异性进行鉴定。结果获得了4株可稳定分泌单克隆抗体的杂交瘤细胞2D3、4E6、1C8和1E5,诱生的腹水效价分别为1∶1×107、1∶1×106、1∶1×105、1∶1×106,亚类鉴定表明2D3为IgG1,其余3株均为IgG2b;特异性鉴定显示它们与多种毒素均无交叉反应,经过亲和层析,获得了纯化的单抗。结论获得了特异性的相思子毒素单克隆抗体,为建立相思子毒素的检测及纯化方法奠定了基础,其中4E6的效价最高,可作为检测相思子毒素的核心试剂。

Preparation,purification and characterization of monoclonal antibodies against abrin

Objective To prepare monoclonal antibodies against abrin toxin and to characterize its properties. Methods BALB/c mice were immunized with the abrin. The splenocytes of the immunized mice were fused with Sp2/0 myeloma cells by hybridoma technique. The antibodies against abrin were screened by indirect enzyme-link immnoadsorption assay (ELISA). The hybridoma cells that secrete antibody were cloned by limiting dilution method. Results Four hybridoma cell lines were obtained. The monoclonal antibodies 2D3 belonged to IgG1 subclass, 4E6, 1C8 and 1E5 belonged to IgG2b subclass. The titers of hybridoma cell induced ascites were 1∶1×107, 1∶1×106, 1∶1×105 and 1∶1×106 respectively. Conclusion Four monoclonal antibodies against abrin were prepared successfully, which will facilitate establishing detection method of abrin. The mAb 2D3 against abrin could be used as the key reagent for establishment of abrin detection method.