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Detection of antigen epitope for bullous pemphigois antigenic protein and the development of autoantibody detection using synthetic polypeptide

大疱性类天疱疮抗原蛋白抗原表位的检测及合成多肽自身抗体检测的发展

基本信息

批准号：
02557045
负责人：
NISHIKAWA Takeji
金额：
$ 6.78万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

The final destination of this developmental scientific research was to detect anti-basement membrane zone (BMZ) antibodies from patients with bullous pemphigoid (BP) using a newly synthesized polypeptide antigen that contains epitope for BMZ antigen protein.To approach this purpose, we have collected as many sera as possible from Japan, U. K. and U. S. patients with BP. We have found that the BMZ antibodies from different continents are similarly heterogenous in terms of antigenic epitope as assessed by immunofluorescence, immunoblotting and immunoprecipitation. Then, we have characterized BP sera by use of recombinant BP antigen proteins and tested the possibility of applying ELISA technique. At the same time we specified the epitope of the"230 kD BP antigen for human anti-BMZ monoclonal antibody 5E by means of deletion mutants of the mouse CDNA Clone that encodes for about carboxyl-terminal half of t. he 230 kD BP antigen protein. The epitope for 5E was found to localize in the 114 amino acid residues encoded by Bgl 11 - StuI fragment of the CDNA. From these studies the following conclusions were obtained ; (1) there are several epitopes present in the BEIZ antigens and (2) more than 3 synthetic peptides are necessary to cover the entire epitopes for ELISA technique to detect 230 kD antigen only. More investigations are needed to reach the final destination.

这项发展性科学研究的最终目的是使用一种新合成的含有BMZ抗原蛋白表位的多肽抗原检测大疱性类天疱疮(BP)患者的抗基底膜带(BMZ)抗体。为了达到这一目的，我们收集了尽可能多的来自日本、英国和美国的BP患者的血清。我们发现，来自不同大陆的BMZ抗体在免疫荧光、免疫印迹和免疫沉淀方面的抗原表位具有相似的异质性。然后，我们用重组BP抗原蛋白对BP血清进行了鉴定，并验证了应用ELISA法的可能性。同时，我们通过小鼠CDNA克隆的缺失突变体，确定了人抗BMZ单抗5E的230kDBP抗原表位，该CDNA克隆体编码约230kDBP抗原蛋白。5E的表位位于CDNA的BGL 11-StuI片段编码的114个氨基酸残基上。通过这些研究，得出以下结论：(1)BEIZ抗原中存在多个表位；(2)用ELISA技术仅检测230kD抗原，需要3个以上的合成肽才能覆盖整个表位。需要进行更多的调查才能到达最终目的地。