CK20单克隆抗体研制及鉴定

Preparation and identification of antiCK20 monoclonal antibody

目的:研制CK20单克隆抗体.方法:采用CK20抗原免疫小鼠,用免疫的脾细胞和SP2/0骨髓瘤细胞通过PEG4000融合,以HT培养基选择培养,间接ELISA法筛选阳性孔和有限稀释法克隆细胞后,接种小鼠腹腔制备腹水抗体,抗体纯化采用以SPA-Sepharose CL-4B为固定相的亲和层析法,最后用双向琼脂扩散法、Western blot和间接ELISA法鉴定抗体的特异性、Ig亚型和效价.结果:获得一株稳定分泌抗CK20单克隆抗体的杂交瘤细胞,染色体众数为101(99～103),其腹水抗体和纯化的抗体效价均为1:106,属IgG1亚型.结论:成功获得了CK20单克隆抗体,命名为L20031030,其特异性强,效价高.

Objective：To produce and identify antiCK20 monoclonal antibody. Methods： Lymphocytes from the spleen of mice being immunonized by CK20 antigen were fused with the myeloma cell line（SP2/0） using PEG4000. Hybridodma cells were established by selective growth of the fusion cells in the HT medium, and the presence of antiCK20 antibody was screened by inderect ELISA, and the clonality was achieved by limiting dilution. We have incubated cloning cells into mouse abdominal cavity to produce ascitic fluid contained monoclonal antibody. Chromatography with SPA-Sepharose CL-4B affinity column were emploied to isolate the monoclonal antibody from ascitie fluid. Finally, the antibody were tested the activities and senfivities, isoforms and titer through Western blot, two directions immuning diffusion of agar and ELISA.Results： Only one hybridoma cell line, seereating MeAb against CK20,had been established. The modal number of chromosome is 101 （99-103）. The results of identifications showed that the antibodies kept high activitis and seusitivitis in detecting sample. The titer of ascitic fluid and the McAb purified are 1 ： 106 equally. The immunoglobulin of the McAb is classified as IgG1. Conclusion： AntiCK20 monoclonal antibody have been produced succesfully with high sensitive and active and was named L20031030.