| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2004: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2003: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
1)Production of an anti-sulfoglycolipid monoclonal antibody: An anti-sulfoglycolipid monoclonal antibody, DI8, was obtained by immunizing glyolipid sulfotransferase-knockout mice with pure sulfatide. The DI8 antibody reacted to sulfatide as well as seminolipid, but not to neutral glycolipids, gangliosides and other sulfated carbohydrate chains. DI8 may recognize the non-reducing terminal Gal 3-sulfate structure, because it did not react to SM2, whose sulfate group is attached to the inner Gal residue. After isolation of the genes of the variable regions of the heavy chain and light chain of DI8 IgG, they were connected through a linker. The resultant scFv gene was expressed in a phage display system. Finally, it was demonstrated that the soluble scFv antibody preserves the specificity of the original DI8 antibody.
2)Production of monoclonal antibodies that recognize molecular complexes including sulfoglycolipids on lipid rafts: Seminolipid was recovered from the detergent insoluble micr
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odomain(DIM) fraction of spermatogenic cells, suggesting that seminolipid is included in lipid rafts of the plasma membranes of spermatogenic cells. Monoclonal antibodies were then generated by immunizing syngenic mice with the DIM fraction of spermatogenic cells. Three clones were selected on the basis that they strongly reacted to DIM, but weakly reacted to seminolipid. All of them were IgM. Two of the three clones reacted to specific protein bands on Western blotting analysis. The other clone (#1) did not react to any bands, suggesting that it may recognize a three-dimensional structure. The #1 Ab -recognizing molecular complex was found to bind to sulfatide but not to galactosylceramide. Identification of the #1 Ab -recognizing molecular complex is underway.
3)Production of an animal model of autoimmune disease against sulfoglycolipids : After immunizing glycolipid sulfotransferase-knockout mouse with sulfatide, and confirming that antiserum activity against sulfatide was raised, spreen cells were isolated from the immunized mouse and transplanted into syngenic wild-type mice. As a result there was no increase of the titer of anti-sulfatide antibody in the recipient mice, and they showed no abnormality. Less
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