HLA作为主要组织相容性抗原在移植免疫起着重要作用，但患者在缺乏抗供者HLA抗体和交叉配型阴性的情况下仍然发生对移植物的排斥反应。前期研究结果提示内皮细胞(EC)特异性抗体由于不被T和B淋巴细胞的交叉配型试验检出，临床上HLA交叉配型反应阴性的肾移植出现不明原因的排斥反应很可能与该EC抗原有关。探索EC抗原的编码基因是当前急需解决的关键问题。我们应用HUVEC细胞模型和细胞流式技术对EC抗体血清样本进行了鉴定，并从血清中提纯该抗体，再通过免疫沉淀直接捕获相应的EC抗原。利用蛋白质组学和质谱分析技术，确定该EC抗原的人体编码基因。随即开展对该基因测序分型，血清学相关性分析和人群该EC基因多态性调查。制备常见等位基因型的重组蛋白质，制成多抗原液态芯片Allo-抗体检测系统。确定新EC移植抗原和检测其抗体特异性为建立器官移植的内皮细胞虚拟配型提供实用的实验数据，有利于预防临床器官移植免疫排斥反应

The waiting for kidney transplantation in our country currently is counted by millions and millions. Among these, over a third or more of them could be sensitized against the polymorphic endothelial allo-antigens. The HLA antigens are the major targets of the immune response against the donor tissue. However, rejection may occur in the absence of antibodies against the HLA antigens of the donor. It has therefore been proposed that other antigens expressed in the graft endothelium, and not normally detectable in peripheral blood lymphocytes, may be additional targets that can cause allograft loss. We and others have used endothelial cells isolated from umbilical veins (HUVEC) to detect antigens that may be responsible for kidney allograft rejection in patients in whom antibodies against HLA cannot be detected even using the most sensitive techniques. These polymorphic endothelial antigens could be a major source of sensitization and a cause of unexplained graft loss in patients with negative HLA crossmatches. Understanding these antigens could have an important impact that could also benefit recipients of other organs such as heart and lung. Presence of such antibodies, in the complete absence of antibodies against HLA, was strongly associated with decreased survival of kidney allografts obtained from deceased donors. We now would like to use immunological and protein identification techniques to recognize and characterize these antigens. Experiments to be performed include: 1). Antibodies to human umbilical cord vein endothelial cells (HUVEC) determined by flow cytometry; 2). Identification of sera with antibodies against polymorphic endothelial antigens. 3). Immunoprecipitation of endothelial cell antigens. 4). Protein identification by mass spectrometry. 5). Once the genes are identified, nucleotide sequencing will be used to map the polymorphisms and to correlate them with the serologic results. 6). The next step will be to produce recombinant antigens representing the polymorphic immunogenic sequences identified, which can be used for the development of an immunoassay based on Luminex bead multiplex technology. Our group has documented experience and published experiments using all of these approaches in connection with the study of MICA antigens and antibodies and their role in organ transplantation. The techniques of single-antigen Luminex bead assays for antibodies against polymorphic antigens are fully developed and operational.Methods, procedures and materials for the various components are ready for deployment immediately when support becomes available. Preliminary evidence suggests that characterization of these endothelial antigens could have an important effect on the success rate of organ transplantation, which would impact the health of many patients with organ failure for whom transplantation is the best treatment option. Our group is in a unque position to solve this problem now.