| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1995: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1994: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
(1) Insulin-dependent diabetes mellitus (IDDM) is an organ-specific autoimmune disease that causes in the destruction of pancreatic islet beta-cell and insulin deficiency. To obtain cDNAs encoding autoantigens recognized by islet specific autoantibodies in IDDM sera, cDNA library was prepared using mRNAs purified from human islet beta-cells. I have cloned full length protein-coding sequences for human GAD (glutamic acid decarboxylase) 65 and 67 from the library. The recombinant human islet GAD 65 protein produced in E.coli could be detected by rabbit polyclonal anti-GAD Abs but not by autoantibodies of IDDM patients. Accordingly we established a mammalian cell line stably producing recombinant human GAD 65 and developed an assay of the IP-Western method (immunoprecipitation followed by immunoblotting using mAb as a first antibody) to detect anti-GAD autoantibodies. This assay showed higher sensitivity and specificity, 83.3% and 100%, respectively, compared with those for islet cell antibodies (ICA), antibodies against 64,000-Mr islet cell proteins (64K antibodies) and antibodies against GAD purified from pig brain. The correlation between GAD 65 antibodies and ICA or 65K antibodies was significant (r=0.60, P=0.0003 and r=0.47, P=0.07, respectively). GAD 65 antibodies and antibodies against GAD purified from pig brain correlated well(r=0.70, P=0.0001). The quantitative IP-Western is highly sensitive and specific in detecting anti-CAD 65 autoantibodies, and thus useful in predicting the development of IDDM in high risk patients.
(2) To obtain other autoantigens recognized by sera of IDDM patients, we screened the lambda gt11 cDNA library derived from human islet beta-cells. We cloned a cDNA encoding protein recognized by IDDM sera and determined its nucleotide sequences. We are planning to produce the recombinant protein encoded by the cDNA in eukaryote cells and establish an assay system to detect a novel autoantibody.
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