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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.

在骨髓和肾脏的器官移植中，人类白细胞抗原的配型是取得良好效果的关键。血清学配型是一种成熟、简便的人类白细胞抗原配型方法。因此，在器官移植中为供受者提供足够的抗HLA抗血清是非常重要的。但抗-HL A抗血清的收集是通过费时费力地筛选孕期妇女血清而获得的，且抗血清的数量受到明显的限制。为了克服这一问题，我们试图通过用人细胞免疫人类白细胞抗原转基因小鼠来制备良好的针对人类白细胞抗原11类分子多态决定簇的单抗。为此，我们成功构建了人类白细胞抗原DRA、DQw4和DQw6转基因小鼠。转基因小鼠脾细胞表达的人类白细胞抗原分子能刺激非转基因小鼠淋巴T细胞发生明确的混合淋巴细胞反应，因此这些转基因小鼠表达的HLA分子具有免疫学功能。2)转基因小鼠获得了对转基因编码的人类白细胞抗原分子的耐受性。3)转基因小鼠对特定可溶性抗原获得或丧失免疫反应性。用表达同种异体HLA11分子的人B淋巴母细胞系免疫转基因小鼠，将免疫脾细胞与小鼠骨髓瘤细胞系P3XAg8通过标准细胞聚乙二醇化融合。用HAT培养液筛选杂交瘤细胞，用ELISA法和流式细胞术检测抗体与细胞组的结合能力，以确定培养上清液产生的单抗的特异性。建立了4种区分DR2亚型的单抗，其获得针对人类白细胞抗原多态决定簇的单抗的频率似乎高于以往用人细胞免疫非转基因小鼠的方法。