权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Production of monoclonal antibody specific to HLA by utilizing HLA transgenic mice.

利用 HLA 转基因小鼠生产 HLA 特异性单克隆抗体。

基本信息

批准号：
01870026
负责人：
NISHIMURA Yasuharu
金额：
$ 2.62万
依托单位：
Medical Institute of Bioreguration, Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1991
项目状态：
已结题

项目摘要

The matching of HLA is very important to obtain good results in organ transplantation of bone marrow and kidney. Serological HLA typing is a well established and easy method for HLA typing. Therefore it is very important to supply enough anti-HLA antisera for HLA typing of donors and recipients in organ transplantation. But anti-HLA antisera are collected by a laborious and time consuming screening of sera from women having child and the amount of antisera was obviously limitted. In order to overcome this problem, we attempted to produce good monoclonal antibodies specific to polymorphic determinants of HLA class 11 molecules by immunizing HLA transgenic mice with human cells. For this aim, we generated HLA-DRA, DQw4 and DQw6 transgenic mice successfully. HLA molecules expressed in these transgenic mice were immunologicaly functional because 1)HLA molecules expressbd on spleen cells of trasgenic mice stimulated definite mixed lymphocyte reaction in lymph node T cells of non-transgenic mouse. 2)Transgenic mice acquired a tolerance to HLA molecules encoded for by transgene. 3)Transgenic mice acquired or lost immune responsiveness to specific soluble antigens. These transgenic mice were immunized intraperitoneally with human B lymphoblastoid cell lines expressing allogeneic HLA class 11 molecules and immune spleen cells were fused with mouse myeloma cell line P3XAg8 by standard cell fusion with polyethylene glycol. Hybridomas were selected by HAT medium and the specificity of monoclonal antibodies produced in culture supernate were determined by an investigation of binding capacity of antibodies to panel of cells utilizing ELISA and flow cytometry. Four monoclonal antibodies which distinguish DR2 subgroups were established and the frequency to obtain mabs specific to polymorphic determinants of HLA molecules seemed to be higher than the previous method in which non-transgenic mice were immunized with human cells.

HLA配型对骨髓、肾等器官移植获得良好效果至关重要。血清学HLA分型是一种成熟且简便的HLA分型方法。因此，为器官移植供、受者的HLA分型提供足够的抗HLA抗血清是非常重要的。但抗-HLA抗血清的制备需要从育龄妇女血清中筛选，费时费力，且抗血清的量明显有限。为了克服这一问题，我们试图通过用人细胞免疫HLA转基因小鼠来产生良好的针对HLA 11类分子多态性决定簇的单克隆抗体。为此，我们成功地建立了HLA-B1、DQw 4和DQw 6转基因小鼠。这些转基因小鼠表达的HLA分子具有免疫学功能，其原因是：1）转基因小鼠脾细胞表达的HLA分子可刺激非转基因小鼠淋巴结T细胞产生一定的混合淋巴细胞反应; 2)转基因小鼠获得了对转基因编码的HLA分子的耐受性。3)转基因小鼠获得或失去对特异性可溶性抗原的免疫应答。用表达同种异体HLA 11类分子的人B类淋巴母细胞系腹腔内免疫这些转基因小鼠，并通过用聚乙二醇的标准细胞融合将免疫脾细胞与小鼠骨髓瘤细胞系P3 XAg 8融合。通过HAT培养基选择杂交瘤，并通过利用ELISA和流式细胞术研究抗体与一组细胞的结合能力来确定培养上清液中产生的单克隆抗体的特异性。建立了四种区分DR 2亚群的单克隆抗体，获得HLA分子多态性决定簇特异性单克隆抗体的频率似乎高于以前用人细胞免疫非转基因小鼠的方法。