The Establishment Of Hybridoma SU-2000 Secreting McAb Against Human Glioma,and The Purification Of The Detected Antigen

TheMcAb against human glioma was first prepared by Schegg in 1981. Because of the existence of blood-brain-barrer and some other reasons, the gene-engineering modified McAb with lower MW and more humanity were produced. However, these modified McAb did not meet the pratical need with undesired quality. Up-to now, preparing McAb though hybridoma is still popular. in this study, the new McAb against human glioma will be prepared still by hybridoma, but with some modification. Part I The preparation of hybridoma secreting monoclonal antibody against human glioma Objective: This study is designed to prepare a new hybridoma producing specific antibody against human glioma. Methods: 1 .The spleen cells of the mice immunized with SHG-44 glioma cells were fused with SP2/O myeloma cells by PEG and cultured with HAT; 2. By ELISA screening, to check out positive clones; 3.By multi-subcloning, to promise all the hybridoma cells secret McAb; 4. by injecting hybridoma cells into the abdomen of mice, acquire ascites containing McAb; 5.The frozen hybridoma cells were lv 4 ~Th!E1t SU-2000 Jrjt~~ revived after half a year. Results: 1. Hybridoma cells grew in 26 wells; 2.One well was checked out as positive; 3. All hybridoma cells were testified to secret McAb; 4.A large amount of ascites was acquired; 5.The frozen hybridoma cells were successfully revived after half ayear. Conclusion: one hybridoma secreting specific monoclonal antibody against SHG-44 glioma cells is preliminarily established, and named SU-2000. Part II Functional and biochemical characterization of monoclonal antibody SU-2000 Objective: To characterize the monoclonal antibody produced by SU-2000 hybridoma for further application. Methods: Assess work included four parts: 1. To make sure the subclass of the McAb SU-2000 by double-way immunodiffusion; 2. The ascites and the purified NIcAb were diluted double by double to assess ti~e McAb抯 immunoreaction with SHG-44 cells; 3. The reaction spectrum of the McAb was checked by immunohistoehemical staining in tissue sections; 4. lmmunofluoreseence and immunohistochemical staining on SHG-44 cells were performed to seek for the localization of the antigen detected by SU-2000 McAb. Results: 1. An imrnunoprecipitation line appeared between SU-2000 McAb and anti-IgGl; 2. The McAb demonstrated positive reaction with all 8 astrocytic gliomas, but negative reaction with non-glioma tumor samples except melanoma. As far as normal human tissue is concerned, no positive reaction happened; 3. Diluted to 1:204800, the V +~A~M~ SU-2000 慽+!~j~4z ascites still showed positive reaction with SHG-44 glioma cells by ELISA; while the purified McAb (1.33mg/mi) showed positive reaction with the dilution of 1:102400; 4. Both immunofluorescence and immunohistochemical staining showed the recognized antigen existing mainly on the cell surface. Conclusion: The monoclonal antibody SU-2000 belongs to IgGlwith high specificity and quuality. Part III The purification of the antigen recognized...