The Establishment Of An Anti-human-osteosarcoma Antibody Producing Hybridoma Cell Lines And The Rudiment Application Of The Anti-body

AIM: Osteosarcoma is the most common malignant tumor of bone and often occur in patients between 12 and 25. It is difficult for treatment and patient who accepted amputation alone often died of pulmonary metastasis in a year. Osteosarcoma is very dangerous especially in children and young adults. Besides operation, adjuvant chemotherapy and radiotherapy, more and more emphasis were play on immunotherapy. The researches of tumor marker and tumor's malignant behavior have been enhanced by the development of technique mono-antibody, and then immunotherapy. In order that we can find the markers of malignant tumor's behavior, we established anti-osteosarcoma antibody producing hybridoma cell lines and to study the characterization of themonoclonal antibody. METHODS: Balb/c mice were immunized with human osteosarcoma cells OS?607 and the immunized spleen cells were fused with SP2/0 cells to raise hybridoma. Using PEG method and limiting dilution, and after screening and cloning , we draw the conclusion that we have built one hybridoma cell line. The positive clones were fused with ELISA and immunohistochemistry methods using OS?607 and normal hepatocytes. The antibody was identified with electrophoresis. Antibody from the positive clones was identified with immunohistochemistry and Western Blot methods. RESULTS: we obtained 12 positive holes, a hybridoma cell line named 3D9 was established from 3 of these positive holes. Who secreted high quality McAbs steadily after 100 days. The McAb 3D9 belong to IgGl subtype. The titer of ascites is 1:1.28X107 and the titer of culture medium is 1:32. With enzyme-labeled immunohistochemical staining on formaldehyde-fixed sections from human osteosarcoma, it was found that the mAbs' corresponding antigens were specifically and highly expressed in human osteosarcoma(83%). Most of them were expressed on the nuclear of cells, no positive expression was observed in kinds of normal tissues. Western-Blot showed 3D9's corresponding molecule weight is Mr54 000. CONCLUSION: A high quality hybridoma is cultured. It may be a new biochemical marker of osteosarcoma, and it's clinical prospect of immunotherapy will be wide. These experiments would be helpful to the future background and clinical works.