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

在骨髓和肾脏器官移植中，HLA的配型对获得良好的移植效果至关重要。血清学HLA分型是一种建立良好且简便的HLA分型方法。因此，在器官移植中，提供足够的HLA抗血清用于供体和受体的HLA分型是非常重要的。但是抗hla抗血清是通过对孕妇血清的筛选来收集的，这是一项费时费力的工作，而且抗血清的数量明显有限。为了克服这一问题，我们试图通过用人细胞免疫HLA转基因小鼠来产生针对HLA 11类分子多态性决定因子的良好单克隆抗体。为此，我们成功地构建了HLA-DRA、DQw4和DQw6转基因小鼠。在这些转基因小鼠中表达的HLA分子具有免疫功能，因为1)在转基因小鼠的脾脏细胞上表达的HLA分子在非转基因小鼠的淋巴结T细胞中刺激了明确的混合淋巴细胞反应。2)转基因小鼠对转基因编码的HLA分子具有耐受性。3)转基因小鼠获得或失去对特定可溶性抗原的免疫应答。将表达同种异体HLA 11类分子的人B淋巴母细胞样细胞系腹腔免疫这些转基因小鼠，并将免疫脾细胞与小鼠骨髓瘤细胞系P3XAg8通过聚乙二醇标准细胞融合融合。用HAT培养基筛选杂交瘤，利用ELISA和流式细胞术检测抗体与细胞的结合能力，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DR2亚群的单克隆抗体，获得针对HLA分子多态性决定因子的单抗的频率似乎高于先前用人细胞免疫非转基因小鼠的方法。

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.