Bolting And Preparation Of Monoclonal Antibody To Membrane Protein Of Hepatoma Carcinoma Cell

Objective: To prepare monoclonal antibody (McAb) to membrane protein of hepatoma carcinoma cell by hybridoma technique.Methods: In this study,we get membrane protein of hepatoma carcinoma cell by grinding,membrane protein extraction kit and hyposmolality as antigen,purify it by gradient centrifugation and quantitate it by Bradford method. Balb/c mice were immunized with membrane protein(25ug-50ug every time) prepared by us. The splenocytes of the immuned mice were fused with myeloma cells by polyethylene glycol and the hybridoma cells were selected in HAT medium and HT medium.Enlarge the culture of the positive hybridomas and prepare McAb.The positive hybridomas secreting McAb to membrane protein of hepatoma carcinoma cell were screened by means of ELISA with membrane protein as coating antigen. The reactivity of McAb secreted by the positive hybridoma to membrane protein was confirmed by means of ELISA methods.Results: In partâ… ,the serum antibody titer of the mice reached 1:105 after the fourth immunization.We got ten positive hybridomas after cell fusion and got five hybridomas after macroculture,but these hybridomas can not secret McAb by means of ELISA methods. In partâ…¡, The serum antibody titer was negtive after the fourth immunization. Then removed the organic solvent by vacuum drying and immuned mice once again,but the serum antibody titer of the mice still remained negtive.The immunization has failed. In partâ…¢,the serum antibody titer of the mice reached 1:106 after the fifth immunization.We got twenty-two positive hybridomas after cell fusion and got fifteen hybridomas after macroculture,but these hybridomas can not secret McAb by means of ELISA methods.Conclusion: There are some defects in preparation monoclonal antibody (McAb) to membrane protein of hepatoma carcinoma cell extracted by grinding or by hyposmolality by hybridoma technique. The purity of membrane protein is low and the quantity is small by the two means.The antigenic element is complex, so it is difficult to prepare McAb to membrane protein of hepatoma carcinoma cells.The immunizing dose of 25ug is insufficient in partâ… ,and it should be increased to 50ug erery time.The antibody secreted in serum is polyclonal.The serum antibody titer to single antigen is lower than polyclonal antibody titer,so we should fuse splenocytes of the immuned mice and myeloma cells when the serum antibody titer reached 1:106.The purity and quantity of membrane protein extracted by kit is better than by grinding and hyposmolality. The organic solvent in kit may have affected antigenicity and vacuum drying can not remove it completely,thus affect immunization result.