Detection of autoantibodies in sera of patients with insulin-dependent diabetes mellitus.

胰岛素依赖型糖尿病患者血清中自身抗体的检测。

基本信息

批准号：
06671040
负责人：
MORIUCHI Ryozo
金额：
$ 1.28万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

(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.

（1）胰岛素依赖性糖尿病（IDDM）是一种特定器官的自身免疫性疾病，会导致胰岛β细胞和胰岛素缺乏症的破坏。为了获得编码由IDDM Sera中胰岛特异性自身抗体识别的自身抗原的cDNA，使用从人类胰岛β细胞中纯化的mRNA制备cDNA库。我从文库中克隆了人GAD（谷氨酸脱羧酶）65和67的全长蛋白质编码序列。兔多克隆抗GAD ABS可以检测到在大肠杆菌中产生的重组人类胰岛65蛋白，而不是由IDDM患者的自身抗体检测。因此，我们建立了稳定产生重组人GAD 65的哺乳动物细胞系，并开发了IP-Western方法的测定法（免疫沉淀，然后使用MAB作为第一种抗体进行免疫印迹）来检测抗GAD自身抗体。与胰岛细胞抗体（ICA）相比，该测定法分别显示出更高的灵敏度和特异性，分别为83.3％和100％，对64,000-MR Islet细胞蛋白（64K抗体）的抗体和针对从猪脑纯化的GAD纯化的抗体。 GAD 65抗体与ICA或65K抗体之间的相关性显着（r = 0.60，p = 0.0003和r = 0.47，p = 0.07）。 GAD 65抗猪脑相关的纯化GAD的抗体和抗体（r = 0.70，p = 0.0001）。定量IP-Western在检测抗CAD 65自身抗体时高度敏感且具有特异性，因此有助于预测高风险患者的IDDM的发展。（2）为获得由IDDM患者识别的其他自身抗原，我们筛选了Lambda GT11 cdna库从人类iSlet islet beta beta beta beta-cells中筛选。我们克隆了由IDDM Sera识别的蛋白质的cDNA，并确定了其核苷酸序列。我们计划生产由真核生物细胞中cDNA编码的重组蛋白，并建立一个检测新型自身抗体的测定系统。