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

我们发现，在类风湿性关节炎的不同器官中淀粉样蛋白沉积中存在一种新的抗原，该抗体可识别一种新的抗原。此外，用24个氨基酸组成的β蛋白合成肽免疫BALB/c小鼠获得的脾细胞与小鼠骨髓瘤X63-Ag8.653细胞融合，产生抗β蛋白的单抗(TB_1、T_2、T_3、B_4和T_5)。这些抗体均与阿尔茨海默病(AD)或老年性痴呆阿尔茨海默型(SDAT)患者脑组织淀粉样斑块和淀粉样血管病变特异结合，而与正常脑或其他组织中的蛋白质完全不结合。甲酸对脑组织的预处理增强了这种反应性。在此条件下，即使在少量斑块、老年斑、冠状淀粉样蛋白和毛细血管淀粉样物沉积中也可见明显的阳性反应。Western印迹分析表明，SDAT脑提取液中单抗TB2和Tb3主要与116kD条带反应。然而，当使用从其他疾病患者的正常脑提取液时，他们未能反应出116kD条带。双向电泳法显示，116kD条带在p8.5左右发生迁移。MoAbTb1、Tb2和Tb4与由亲水性氨基酸组成的β蛋白合成肽(24个氨基酸)的第一部分(N端附近的7个氨基酸)发生强烈反应。

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.