维普中文期刊产品整合服务

Preparation of monoclonal antibody against apoptosis-associated antigens of hepatoma cells by subtractive immunization

查看全文 作　　者：Lian-Jun Yang Wen-Liang Wang Department of Pathology,Institute of Cancer Research,The Fourth Military Medical University (FMMU),Xian 710032,Shaanxi Province,China 高影响力作者 出　　处：《World Journal of Gastroenterology》索引2002年第8卷第5期,共7页高影响力期刊 基　　金：Army Science Foundation,No.98M111 摘　　要：AIM: To elucidate the expression of the apoptosis-associatedmolecules in human primary hepatocellular carcinoma (HCC)cells, and prepare the monoclonal antibodies (mAb) againstthe apoptosis-associated antigens of HCC cells.METHODS: Human HCC cell line HCC-9204 cells wereinduced apoptosis with 60 mL.L-1 ethanol for 6 h and theirmorphological changes were observed by transmissionelectron microscope. The cell DNA fragmentations weredetected by Terminal Deoxynucleotidyl transferase-mediateddUTP nick end labeling (TUNEL) assay, and the cell DNAcontents by flow cytometry. Ten mice were immunized withethanol-induced apoptotic HCC-9204 cells with the methodof subtractive immunization, while the other 10 mice usedas the control were immunized by the routine procedures.The tail blood of all the mice were prepared after the lastimmunization, and the produced antibodies were determinedby the immunocytochemical ABC staining. The splenic cellsof the mice whose tail blood sera-HCC-9204 cells serumreactions were most different between the apoptotic andthe non-apoptotic were prepared and fused with the mousemyeloma cell line SP2/0 cells. The positive antibodies wereselected by ELISA assay. The fusion rates of hybridoma cellsand the producing rates of antibodies were calculated. Thefused cells that secreted candidate objective antibody werecloned continually with the of limited dilution method, andthen selected and analyzed further by theimmunocytochemical ABC staining. The chromosomes of thecloned hybridoma cells that secreted objective mAb and themAb immunoglobulin (Ig) subtype of the prepared mAb werealso determined. The molecular mass of the mAb associatedantigen was analyzed by Western blot assay.RESULTS: HCC-9204 cells treated with 60 mL.L-1 ethanolfor 6 h, manifested obvious apoptotic morphological changes,the majority of the cells were TUNEL-positive, and the sub-G1 apoptotic peak was evident. There were 2 mice in theexperimental group whose tail blood serum reacted stronglywith the apoptotic HCC-9204 cells, but weakly with theirnon-apoptotic counterparts. In the fusion rates of hybridomacells as well as the producing rates of the antibody deseribedabove, there did not show significant difference betweenthe experimental and the control group, but weakly withnon-apoptotic HCC-9204. However, the total producing rateof antibodies in the experimental group was significantlylower compared with the control (P<0.01), and so was theproducing rate of the antibodies which reacted strongly withboth apoptotic and non-apoptotic HCC-9204 cells(P<0.01).After cloned continually for several times the cell that producemAb which reacted strongly with the nuclei of ethanol-induced apoptotic HCC-9204 cells, but very weakly with thatof non-apoptotic cells was selected out. Chromosome analysisrevealed that the selected cell was with the universalcharacteristics of the monoclonal hybridoma cells whichsecreted mAb, and the Ig subtype of the prepared mAb wasIgG1. The molecular mass of this mAb associated antigenof was about 75 ku.CONCLUSION: Subtractive immunization is a useful methodto prepare the mAb against the apoptosis-associated antigensof cells. The expression of some molecules increases to someextent in HCC-9204 cells in the process of apoptosis inducedby Iow-concentration ethanol. The mAb that may be againstethanol-induced apoptosis-associated antigens of HCC cellswas successfully prepared and primarily identified. 关 键 词：肝癌 单克隆抗体 免疫抑制 细胞凋亡相关抗原
相关文献

参考文献(66)

引证文献(15)

耦合文献(560)

网站首页 | 关于我们 | 联系我们 | 产品服务 | 客服中心 | 广告服务 | 版权声明 | 网站联盟 | 友情链接 | 售卡网点

版权所有© 渝B2-20050021-1　渝公网安备 50019002500403号　违法和不良信息举报中心

互联网出版许可证 新出网证(渝)字10号 全国400电话 - 免长途话费