Tuso P J, Cramer D V, Middleton Y D, Kearns-Jonker M, Yasunaga C, Cosenza C A, Davis W C, Wu G D, Makowka L
Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California.
Transplantation. 1993 Sep;56(3):651-5. doi: 10.1097/00007890-199309000-00029.
Human-to-pig xenoantibodies may constitute a major obstacle to the successful use of pigs as xenograft donors for human transplantation. Our studies demonstrate that normal human serum contains antibodies, primarily IgM, that are cytotoxic for pig aortic endothelial cells (PAECs). These antibodies bind to several antigens isolated from PAECs, lymphocytes, platelets, red blood cells, and the kidney. Absorption of human serum with pig lymphocytes removes the cytotoxic activity to PAECs and some, but not all, of the IgM antibodies capable of binding in an ELISA assay to the PAECs. The cytotoxic antibodies are inactivated by 2-mercaptoethanol, suggesting that they are primarily IgM. Whole cell extracts of PAEC, lymphocytes, platelets, red blood cells, and kidney were prepared and analyzed by Western blots to establish the cellular distribution of the xenoantigens that react with human IgM in pooled human serum. Results showed that several of the most intensely stained bands migrated between 24 and 66 kDa. High molecular weight bands (> 100 kDa) were observed only in kidney, platelet, and PAEC preparations. Human IgM xeniantibodies also reacted strongly in Western blots to endothelial cell membranes proteins with molecular weights of 62, 48, 42, 36, 34, 28, and 26 kDa. Absorption of human serum with pig lymphocytes removes IgM binding to all bands except for a 34-kDa Treatment of the PAEC membrane proteins with proteinase K disrupts the binding of the human IgM antibodies. Similar treatment with glycosidase F) resulted in a decrease in molecular weight of the 28- and 26-kDa bands, suggesting that these xenoantigens are glycoproteins and that antibody binding to some xenoantigens may not require glycosylation.
人源抗猪异种抗体可能是猪作为人类移植异种移植物供体成功应用的主要障碍。我们的研究表明，正常人血清中含有主要为IgM的抗体，这些抗体对猪主动脉内皮细胞（PAEC）具有细胞毒性。这些抗体与从PAEC、淋巴细胞、血小板、红细胞和肾脏中分离出的几种抗原结合。用猪淋巴细胞吸收人血清可消除对PAEC的细胞毒性活性以及一些（但不是全部）在ELISA检测中能够与PAEC结合的IgM抗体。细胞毒性抗体可被2-巯基乙醇灭活，表明它们主要是IgM。制备了PAEC、淋巴细胞、血小板、红细胞和肾脏的全细胞提取物，并通过蛋白质印迹法进行分析，以确定与混合人血清中的人IgM反应的异种抗原的细胞分布。结果显示，几条染色最深的条带迁移在24至66 kDa之间。仅在肾脏、血小板和PAEC制剂中观察到高分子量条带（>100 kDa）。人IgM异种抗体在蛋白质印迹中也与分子量为62、48、42、36、34、28和26 kDa的内皮细胞膜蛋白强烈反应。用猪淋巴细胞吸收人血清可消除IgM与除34 kDa以外所有条带的结合。用蛋白酶K处理PAEC膜蛋白会破坏人IgM抗体的结合。用糖苷酶F进行类似处理会导致28 kDa和26 kDa条带的分子量降低，表明这些异种抗原是糖蛋白，并且抗体与某些异种抗原的结合可能不需要糖基化。
