氯霉素单克隆抗体的制备与纯化

Preparation and purification of monoclonal antibodies against chloramphenicol

目的制备氯霉素(CAP)单克隆抗体。方法用人工合成抗原氯霉素-牛血清白蛋白(CAP-BSA)免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0按5∶1的比例融合,间接竞争ELISA法和有限稀释法进行单克隆杂交瘤细胞的筛选;制备腹水抗体;采用HiTrap rProtein A FF亲和色谱柱纯化抗体,用间接竞争ELISA法和间接ELISA测定抗体特异性。结果得到两株能稳定分泌单克隆抗体的杂交瘤细胞株,细胞培养上清中抗体效价达10-4以上,腹水抗体效价达10-7以上,纯化后的单克隆抗体纯度达98%,回收率达80%,抗体活性好并且与BSA、甲砜霉素、磺二甲基嘧啶等无交叉反应。结论成功获得两株能稳定分泌单克隆抗体的杂交瘤细胞,对动物性食品中CAP的检测具有较大的价值。

Purpose To prepare monoclonal antibody against chloramphenicol （CAP）. Methods BALB/c mice were immunized with CAP-BSA,and spleen cells from the mice were fused with myeloma aells SP2/O at ratio of 5：1. Monoclonal hybridomas were screened by competitive indirect enzyme-linked- [mmunoadsorbent assay（ciELISA） and limited dilution. Mouse ascites were prepared by intraperitoneal in- jection of hybridoma cells. The ascites were purified by protein A affinity chromatography. Specificity and activity of the ascites antibody were determined by ciELISA and indirect ELISA. Results Two strains of hybridoma cell lines obtained could secrete McAb stably. The titre of MAb of cell culture supernate is nlore than 10-4 ,and the titre of prepared ascites MAb is more than 10-7. After purification,the purity of Lhe obtained monoelonal antibody was more than 98% and recovery reached 75%. There is no cross-activ- ity between McAb against chloramphenicol and some compounds. Conclusion The two obtained strains of hybridoma cell lines can secrete McAb stably. The monoclonal antibodies can be used in the future re- search.