Development of a consensus protocol to quantify primate anti-non-Gal xenoreactive antibodies using pig aortic endothelial cells.
Development of a consensus protocol to quantify primate anti-non-Gal xenoreactive antibodies using pig aortic endothelial cells.
复制标题
使用猪主动脉内皮细胞来量化灵长类动物抗非洲 - 糖异种反应性抗体的共识方案的开发。
DOI：
10.1111/xen.12125
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发表时间：
2014-11
影响因子：
3.9
通讯作者：
Pierson RN 3rd
中科院分区：
文献类型：
--
作者：
Azimzadeh AM;Byrne GW;Ezzelarab M;Welty E;Braileanu G;Cheng X;Robson SC;McGregor CG;Cooper DK;Pierson RN 3rd
在异种移植领域工作的科学家没有使用统一的方法来测量和报告对非半乳糖α(1，3)半乳糖(非半乳糖)表位的自然和诱导抗体反应。在移植带血管的GalTKO猪器官后，这种体液反应被认为是特别致病的，拥有更统一的检测和报告格式将极大地促进实验室之间的比较。流式细胞术可以检测抗体对天然位置的完整抗原的反应性和细胞膜上的构象。我们建立了一种简便、重复性好的流式细胞术检测非Gal型猪抗原抗体的方法，该方法以野生型和α-1，3-半乳糖基转移酶基因敲除猪的原代猪主动脉内皮细胞和经细胞培养适应的猪主动脉内皮细胞为材料，建立了一种检测非Gal型猪抗原抗体的流式细胞术。我们在这里提出的共识方案是基于四个异种移植中心常规使用的程序，并在三个地点使用共享的细胞和血清样本进行了独立评估。我们的观察结果支持使用经GalTKO PAEC KO：15502细胞适应的细胞培养作为常规方法来检测人和狒狒血清中抗非半乳糖抗体的反应性。总之，我们开发了一种检测方法，能够以可靠和一致的方式检测人类或狒狒血清中存在的天然和诱导的非半乳糖异种反应性抗体。这种用于报告数据的共识分析和格式应该是大多数实验室都可以访问的，并且对于评估多个研究中心之间的实验结果是有用的。采用这种分析方法和报告数据的格式应有助于检测、监测和详细描述非半乳糖抗体反应。
Scientists working in the field of xenotransplantation do not employ a uniform method to measure and report natural and induced antibody responses to non-Galα(1,3)Gal (non-Gal) epitopes. Such humoral responses are thought to be particularly pathogenic after transplantation of vascularized GalTKO pig organs and having a more uniform assay and reporting format would greatly facilitate comparisons between laboratories. Flow cytometry allows examination of antibody reactivity to intact antigens in their natural location and conformation on cell membranes. We have established a simple and reproducible flow cytometric assay to detect antibodies specific for non-Gal pig antigens by using primary porcine aortic endothelial cells (pAECs) and cell culture adapted pAEC cell lines generated from wild type and α1,3galactosyl transferase knockout (GalTKO) swine. The consensus protocol we propose here is based on procedures routinely used in four xenotransplantation centers, and was independently evaluated at three sites using shared cells and serum samples. Our observation support use of the cell culture adapted GalTKO pAEC KO:15502 cells as a routine method to determine the reactivity of anti-non-Gal antibodies in human and baboon serum. In conclusion, we have developed an assay that allows the detection of natural and induced non-Gal xenoreactive antibodies present in human or baboon serum in a reliable and consistent manner. This consensus assay and format for reporting the data should be accessible to most laboratories and will be useful for assessing experimental results between multiple research centers. Adopting this assay and format for reporting the data should facilitate the detection, monitoring, and detailed characterization of non-Gal antibody responses.