1. The Establishment Of Two Anti-human-osteosarcoma Antibody Producing Hybridoma Cell Lines And The Characterization Of The Monoclonal Antibodies 2. Purification And Amino Acid Sequence Analysis Of MAb5D3's Corresponding Antigen

AIM: To establish anti-osteosarcoma antibody producing hybridoma cell lines and study the characterization of the monoclonal antibodies. METHODS: The BALB/C mice was immunized with human osteosarcoma cells OS-9607 and the immunized spleen cells were fused with SP2/0 cells to raise hybridomas after three times inoculation. The positive clones of hybridoma were screened with ELISA and immunochemistry methods using OS-9607 and normal Hepatocelluar cells seeded 96-well plate and antibodies from the positive clones were further identified with the immunohistochemistry and Western-Blot methods. RESULTS: There were two hybridoma cell lines be established and both of them, 6E7 and 5D3, secrete high quality mAbs steadily. The mAb6E7 and mAb5D3 belong to IgG1 and IgG2a subtype. The titer of ascetics is 1:10~5 and 1:10~6. 6E7 and 5D3 cells have all the characters of hybridoma. The mAbs' corresponding antigens were specifically and highly expressed in human osteosarcoma. With enzyme-labeled immunohistochemical staining on formaldehyde-fixed sections from human osteosarcoma, it was found that more than 80 percent specimens expressed the corresponding antigen, with no one of ten other kind normal tissues positive. Western-Blot study showed that the corresponding antigens of mAb6E7 and mAb5D3 were a small peptide and a 43KD weight molecule. CONCLUSION: Two high quality hybridomas were cultured and the mAbs secreted by the two hybridomas have osteosarcoma specificity. Further...