Detection of Novel Amyloid-Associated Protein by Monoclonal Antibody and its Gene Analysis

新型淀粉样蛋白相关蛋白的单克隆抗体检测及其基因分析

基本信息

批准号：
01570363
负责人：
IMAI Kohzoh
金额：
$ 1.41万
依托单位：
Sapporo Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

We have found that monoclonal antibody AM34 (IgGl) recognizes a novel antigen which exists in amyloid deposits in various organs of the rheumatoid arthritis.In addition, anti-beta-protein monoclonal antibodies (TB1, TB2, TB3, TB4 and TB5) were generated from the fusion of mouse myeloma X63-Ag8.653 cells with splenocytes obtained from a BALB/c mouse that had been immunized with a synthetic peptide of beta-protein consisting of 24 amino acids. All these antibodies bound specifically to the amyloid changes of senile plaque and amyloid angiopathy of the brain tissues of patients with Alzheimer's disease (AD) or with senile dementia Alzheimer type (SDAT) , but not at all to protein in the normal brain or other tissues by the immunoperoxidase method. Formic acid pretreatment of brain tissue enhanced this reactivity. Positive reactions were clearly visible even in small deposits of plaque, senile plaque crown amyloid and amyloid deposits of capillary vessels under this condition.Western blot analysis revealed that monoclonal antibodies TB2 and TB3 reacted mainly with 116 kD band when extract of SDAT brain was used. They failed to react, however, with 116kD band when extract of normal brain derived from patients with other diseases was used. Two dimensional electrophoresis revealed that the 116 kD band migrated at around pl8.5. MoAbs TB1, TB2 and TB4 strongly reacted with the 1st part (7 amino acids near to N-terminus) of the synthetic peptide (24 amino acids) of the beta-protein, which consists of hydrophilic amino acids.

我们发现单克隆抗体 AM34 (IgGl) 识别存在于类风湿性关节炎各个器官的淀粉样沉积物中的新抗原。此外，产生了抗 β-蛋白单克隆抗体 (TB1、TB2、TB3、TB4 和 TB5)来自小鼠骨髓瘤 X63-Ag8.653 细胞与从 BALB/c 小鼠获得的脾细胞的融合，该小鼠已用 β 蛋白合成肽进行免疫由24个氨基酸组成。所有这些抗体都与阿尔茨海默病（AD）或老年痴呆阿尔茨海默型（SDAT）患者脑组织的老年斑和淀粉样血管病的淀粉样蛋白变化特异性结合，但与正常大脑或其他组织中的蛋白质完全不结合采用免疫过氧化物酶法。脑组织的甲酸预处理增强了这种反应性。在此条件下，即使是小斑块沉积物、老年斑冠淀粉样蛋白沉积物和毛细血管淀粉样蛋白沉积物也能清晰地看到阳性反应。Western blot分析显示，当使用SDAT脑提取物时，单克隆抗体TB2和TB3主要与116 kD条带发生反应。然而，当使用来自其他疾病患者的正常大脑提取物时，它们未能与 116kD 条带发生反应。二维电泳显示116 kD条带在pl8.5附近迁移。 MoAb TB1、TB2 和 TB4 与 β 蛋白合成肽（24 个氨基酸）的第一部分（靠近 N 末端的 7 个氨基酸）发生强烈反应，该部分由亲水性氨基酸组成。