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)胰岛素依赖型糖尿病(IDDM)是一种器官特异性自身免疫性疾病，导致胰岛β细胞破坏和胰岛素缺乏。为了获得编码IDDM血清中胰岛特异性自身抗体识别的自身抗原的cDNA，使用从人胰岛β细胞纯化的mRNA制备cDNA文库。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。大肠杆菌产生的重组人胰岛GAD 65 蛋白可以被兔多克隆抗GAD 抗体检测到，但不能被IDDM 患者的自身抗体检测到。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（免疫沉淀，然后使用 mAb 作为第一抗体进行免疫印迹）检测抗 GAD 自身抗体。与胰岛细胞抗体 (ICA)、针对 64,000-Mr 胰岛细胞蛋白的抗体（64K 抗体）和从猪脑中纯化的针对 GAD 的抗体相比，该检测显示出更高的灵敏度和特异性，分别为 83.3% 和 100%。 GAD 65 抗体与 ICA 或 65K 抗体之间存在显着相关性（分别为 r=0.60，P=0.0003 和 r=0.47，P=0.07）。 GAD 65抗体与猪脑纯化的GAD抗体相关性良好(r=0.70，P=0.0001)。定量IP-Western在检测抗CAD 65自身抗体方面具有高度敏感性和特异性，因此可用于预测高危患者IDDM的发展。(2)为了获得IDDM患者血清识别的其他自身抗原，我们筛选了lambda gt11 cDNA 文库源自人胰岛 β 细胞。我们克隆了 IDDM 血清识别的 cDNA 编码蛋白，并确定了其核苷酸序列。我们计划在真核细胞中生产由cDNA编码的重组蛋白，并建立检测系统来检测新型自身抗体。