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Making the antibody library from pathologic lesion of intestinal bowel disease, and analysis and application of antibody and antigen.

肠道疾病病理病变抗体库的构建及抗体、抗原的分析与应用。

基本信息

批准号：
17390106
负责人：
TSUTSUMI Yutaka
金额：
$ 6.98万
依托单位：
FUJITA HEALTH UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

The purpose of our research is to find useful antibodies and antigens for analyzing the pathogenesis and for diagnosing and treating inflammatory bowel diseases (IBDs). In the lesions of IBDs, numbers of antibody-producing plasma cells infiltrate. However, the antigens recognized by antibodies produced in these immune cells remain unclear. Theoretically, the antibodies locally secreted within the lesions must be closely correlated with the disease process. We thus planned a research directly analyzing the antibodies produced by the plasma cells infiltrating in the lesion.In the first year, we examined the clonality of cDNA encoding antibodies in mouse inflammatory colonic lesions experimentally provoked by dextran sodium sulfate administration, which represents a mouse model of ulcerative colitis. We identified clonalities of the antibody-producing cells infiltrating within the colonic lesion. Therefore, we assumed that the clonality of cDNA encoding antibodies might be useful as crite … More ria for identifying lesion-specific antibodies.In the second year, we analyzed the clonality of antibody cDNA in regional lymph nodes obtained from horseradish peroxidase (HRP)-immunized rats. The HRP reactivity of single chain Fv (scFv) monoclonal antibodies synthesized from nodal tissue cDNA was pursued. We demonstrated clonalities in the nodal tissue cDNA encoding both the heavy and light chains. However, scFv monoclonal antibodies randomly synthesized from the cDNA lacked binding with HRP in the ELISA system. In order to identify cDNA encoding anti-HRP antibodies in the lymph nodes, we screened the cDNA library made from the immunized nodal tissue. Only one type of antibody clone was observed. By random sequence analysis, the heavy chain cDNA of this anti-HRP monoclonal antibody corresponded to one of the several clones. However, the light chain cDNA of the anti-HRP antibody was identical to the sequence that did not show any clonality. These results suggest to our regret that cDNA clones encoding antibody molecules do not necessarily indicate the lesion-specific antibodies. To overcome this difficulty, further analysis is now continuing. Less

本研究的目的是寻找对炎症性肠病（IBD）的发病机制分析、诊断和治疗有用的抗体和抗原。在IBD的病变中，大量产生抗体的浆细胞浸润。然而，这些免疫细胞中产生的抗体识别的抗原仍不清楚。从理论上讲，病变内局部分泌的抗体必然与疾病过程密切相关。因此，我们计划直接分析浸润在病变中的浆细胞产生的抗体的研究。第一年，我们在小鼠溃疡性结肠炎模型中检测了由葡聚糖硫酸钠给药引起的小鼠炎症性结肠病变中编码抗体的cDNA的克隆性。我们确定了抗体产生细胞的克隆浸润在结肠病变。因此，我们假设编码抗体的cDNA的克隆性可能是有用的， ...更多信息在第二年，我们分析了辣根过氧化物酶（HRP）免疫大鼠获得的局部淋巴结中抗体cDNA的克隆性。研究了从结组织cDNA合成的单链抗体（scFv）的HRP反应性。我们证明了编码重链和轻链的结组织cDNA中的克隆性。然而，从cDNA随机合成的scFv单克隆抗体在ELISA系统中缺乏与HRP的结合。为了在淋巴结中鉴定编码抗HRP抗体的cDNA，我们从免疫的淋巴结组织中筛选cDNA文库。仅观察到一种类型的抗体克隆。通过随机序列分析，该抗HRP单克隆抗体的重链cDNA对应于几个克隆之一。然而，抗HRP抗体的轻链cDNA与不显示任何克隆性的序列相同。遗憾的是，这些结果提示编码抗体分子的cDNA克隆并不一定表示病变特异性抗体。为了克服这一困难，目前正在继续进行进一步的分析。少