Screening tumor surface antigen by monoclonal antibody preparation and immunoprecipitation
- VernacularTitle:肿瘤细胞表面抗原的单克隆抗体-免疫沉淀筛选法
- Author:
Xiaoguang XU
;
Haiyan HUANG
;
Chaojun SONG
- Publication Type:Journal Article
- Keywords:
antibodies, monoclonal;
immunoprecipitation;
blotting, western;
antigens, neoplasm
- From:
Medical Journal of Chinese People's Liberation Army
2001;0(11):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To screen the tumor specific antigens, and explore new methods for diagnosis and therapy of tumor. Methods Hela cells, the human cervical carcinoma cell line, were used to immunize the BALB/C mice, and the positive hybridomas were screened by rosette formation screening system in human carcinoma cell line Colo205, Burkitt lymphoma cell line, Raji, etc. The immunofluorescence staining and flow cytometry analysis were performed to characterize if the prepared monoclonal antibodies (mAb) could reorganize the natural membrane antigen expressed on Hela, Colo205, Raji, PLA801, 7901, Jurkat, 293T, ECV304 and resting or activated human peripheral blood mononuclear cells (PBMC). Furthermore, the membrane protein of Hela, Raji and Sp2/0 cells was segregated by ultracentrifugation and the mAbs were cross-linked with the Sepharose CL-4B. In order to identify the molecules which could be recognized by the prepared mAbs, immunoprecipitation was performed to purify the tumor antigen candidates with the cell membrane protein from Hela, Raji and SP2/0 (as negative control) through Sepharose CL-4B cross-linked with the prepared mAbs. Finally, western blotting was performed to detect the molecular weight of the products from the immunoprecipitation. Results Three mAbs were obtained which could recognize the tumor antigen candidates expressed in several human carcinoma cell lines, such as Hela, Raji and Colo205, and some embryonic cell lines, such as 293T and ECV304, but they could not bind to Jurkat, PLA801, 7901, SP2/0, resting or activated human PBMC. Western blotting results indicated that the three mAbs could recognize a 47kD molecule which expressed in Hela and Raji but not on SP2/0 cell membrane. Conclusion Three mAbs, which can recognize tumor membrane antigen candidates, are obtained, providing useful tools for screening and identify the tumor specific antigens.
