Sumitran-Karuppan S, Möller E
Department of Clinical Immunology, Huddinge Hospital, Sweden.
Transplantation. 1994 Sep 27;58(6):713-9.
HLA class I and II antigens were purified to be used for the determination of the specificity of lymphocyte reactive antibodies in renal transplant patients. Purification of HLA antigens was achieved by affinity chromatography using rabbit antibodies directed to human beta 2 microglobulin, W6/32 antibodies that recognize a nonpolymorphic determinant on HLA class I molecules, and BU25 monoclonal antibodies directed to a monomorphic determinant on HLA class II molecules. Pooled platelets and spleen lymphocytes from a large number of donors were used as a source of class I and II antigens. HLA antigen preparations, highly enriched as shown in Western blot experiments, were obtained that caused a dose-dependent inhibition of the binding as well as of the cytotoxicity of W6/32 and BU25 antibodies. The HLA class I preparation did not inhibit the binding of monoclonal antibodies to T or B cell-specific surface markers or to HLA class II antigens. Similar kinds of results were obtained with the HLA class II antigen preparation, which only specifically blocked the class II antibody binding. The antigen preparations were tested for their ability to block binding of antibodies in sera from alloimmunized patients. In order to avoid complications by soluble HLA antigens, all sera were absorbed in ELISA plates coated with anti-class I and anti-class II monoclonal antibodies. The HLA class I and class II antigen preparations were found to be HLA-specific. All data were compared with the conventional method to determine HLA specificity--i.e., specific blocking of class I and class II reactivity using monoclonal antibodies and unabsorbed sera. Soluble HLA antigens, added directly to mixtures of patient sera and lymphocytes used in the cytotoxicity or binding tests, can therefore be used for determination of the presence of HLA antibodies in sera of alloimmunized patients.
纯化 HLA I 类和 II 类抗原，用于测定肾移植患者淋巴细胞反应性抗体的特异性。通过亲和层析法纯化 HLA 抗原，所用的抗体包括：针对人β2微球蛋白的兔抗体、识别 HLA I 类分子上非多态性决定簇的 W6/32 抗体，以及针对 HLA II 类分子上单一型决定簇的 BU25 单克隆抗体。来自大量供体的混合血小板和脾淋巴细胞用作 I 类和 II 类抗原的来源。通过蛋白质印迹实验表明，获得了高度富集的 HLA 抗原制剂，其可导致 W6/32 和 BU25 抗体的结合及细胞毒性呈剂量依赖性抑制。HLA I 类制剂不抑制单克隆抗体与 T 或 B 细胞特异性表面标志物或 HLA II 类抗原的结合。HLA II 类抗原制剂也得到了类似结果，其仅特异性阻断 II 类抗体的结合。检测了抗原制剂阻断同种免疫患者血清中抗体结合的能力。为避免可溶性 HLA 抗原引起的并发症，所有血清均在包被有抗 I 类和抗 II 类单克隆抗体的 ELISA 板中进行吸附。发现 HLA I 类和 II 类抗原制剂具有 HLA 特异性。所有数据均与测定 HLA 特异性的传统方法进行比较，即使用单克隆抗体和未吸附血清特异性阻断 I 类和 II 类反应性。因此，直接添加到细胞毒性或结合试验中所用患者血清和淋巴细胞混合物中的可溶性 HLA 抗原，可用于测定同种免疫患者血清中 HLA 抗体的存在情况。
