| Budget Amount *help |
¥4,400,000 (Direct Cost: ¥4,400,000)
Fiscal Year 1989: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1988: ¥3,800,000 (Direct Cost: ¥3,800,000)
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| Research Abstract |
The object of this project is to produce a panel of monoclonal antibodies that react with human and rat osteoblasts, and to characterize the nature of the antibodies.
Production and characterization of monoclonal antibodies against human osteoblasts Human osteosarcoma cells, Saos-2, were injected intraperitoneally to BALB/C mice 3 times. Spleen cells of those mice were fused with mouse myeloma cells using polyethylen glycol. First screening was performed against cultured Saos-2 cells by an indirect immuno-peroxidase technique. Positive hybridomas in the first screening were re-screened against human osteoblast on the frozen sections of human fetal bones. From these screenings, 4 interesting hybridomas (HOB-1, HOB-2, HOB-3 and HOB-4) were selected for the further analyses. Both HOB-1 and HOB-2 reacted intracytoplasmic antigens in Saos-2 cells and in situ osteoblasts, and occasional cells in spleen, liver and skin fibroblasts. HOB-1 and HOB-2 recognized 59 KD and 57KD species, respectivel
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y, on the Western blotting analysis. HOB-3 recognized cell surface antigens on Saos-2 cells and osteoblasts. The antigen detected by HOB-3 was confirmed to be alkaline phosphatase which belongs to the isoenzyme of bone-liver- kidney type by immunoprecipitation and Western blotting analysis. HOB-4 reacted strongly with extracellular matrix distributed at perlosteal and subcutaneous regions.
Production and characterization of monoclonal antibodies that react with rat osteoblast Rat osteoblastic cell line(ROB-C26) and osteosarcoma cell line(UMR-106) were used for immunization. Four interesting antibodies were selected from hybridomas. ROB232/2 recognized intracytoplasmic antigens in the osteoblast, capillary cells and skin fibroblasts. UP2/96E reacted with osteoblasts and some,bone marrow cells and fibroblasts. UP2/101C showed no reactivity with osteoblasts but strong reactivity with chondrocytes and blood vessels. UP2/73C recognized an intracytoplasmic antigen in muscle cells, but not osteoblasts.
Results of this study suggests that our monoclonal antibodies are useful tools for studying the osteoblast differentiation and function. Less
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