Preparation And Characterization Of The Monoclonal Antibodies Against Human CD133

Objective:This study was to prepare mouse anti-human CD133monoclonalantibody and identification of its biological activity, to provide an initialbasis for the subsequent preparation of anti-human CD133single-chainantibody and the further study of tumor stem cells.Method:BALB/c mice were immunized with purified CD133antigen. Wefused myeloma SP2/0cells with spleen cells of the immunized mice byusing PEG promoting fusion technique. Hybridoma were filtered by ELISA,and the ones that produce monoclonal antibodies were cultured. At thesame time, we tested the activity of hybridoma and the subtypes ofantibody repeatedly. After expanding culture, the positive clones werefrozen and conserved. At last, we tested the binding situation ofanti-CD133monoclonal antibody and CD133+cells in CHO cells andHep-2cells.Result:We successfully fused hybridoma in8blocks of96-well culture platewith the application of PEG-mediated cell fusion, and the rate of fusionwas approximately51.3percent. With continuous cloning culture, we obtained a hybridoma cell lines stably secreting specific anti-CD133molecules(CD133, clone No.15) which was continuously subcultured invitro. The monoclonal antibody was identified by the rapid qualitative teststrips that it was the subtype IgG1, light chain k. With the flow cytometry,we successfully prepared the binding of monoclonal antibody and theCD133antibody in CHO cells and Hep-2cells, and the binding rate wasrespectively78.62percent±8.25percent and1.8percent±0.45percent.Conclusion:We successfully prepared hybridoma cell lines that secreteanti-human CD133monoclonal antibody which was able to be specificallyaffinitive with CD133cells. Monoclonal antibody obtained in this studylays the foundation for the preparation of anti-human CD133single-chainantibody and the construction of immunotoxin targeting laryngeal cancerstem cells.