ATZ11 Recognizes Not Only Z-α1-Antitrypsin-Polymers and Complexed Forms of Non-Z-α1-Antitrypsin but Also the von Willebrand Factor

AimsThe ATZ11 antibody has been well established for the identification of α1-anti-trypsin (AAT) molecule type PiZ (Z-AAT) in blood samples and liver tissue. In this study, we systematically analyzed the antibody for additional binding sites in human tissue.Methods and ResultsUltrastructural ATZ11 binding was investigated immunoelectron microscopically in human umbilical vein endothelial cells (HUVECs) and in platelets of a healthy individual. Human embryonic kidney (HEK293) cells were transiently transfected with Von Willebrand factor (VWF) and analyzed immunocytochemically using confocal microscopy and SDS-PAGE electrophoresis followed by western blotting (WB). Platelets and serum samples of VWF-competent and VWF-deficient patients were investigated using native PAGE and SDS-PAGE electrophoresis followed by WB. The specificity of the ATZ11 reaction was tested immunohistochemically by extensive antibody-mediated blocking of AAT- and VWF-antigens.ATZ11-positive epitopes could be detected in Weibel-Palade bodies (WPBs) of HUVECs and α-granules of platelets. ATZ11 stains pseudo-WBP containing recombinant wild-type VWF (rVWF-WT) in HEK293 cells. In SDS-PAGE electrophoresis followed by WB, anti-VWF and ATZ11 both identified rVWF-WT. However, neither rVWF-WT-multimers, human VWF-multimers, nor serum proteins of VWF-deficient patients were detected using ATZ11 by WB, whereas anti-VWF antibody (anti-VWF) detected rVWF-WT-multimers as well as human VWF-multimers. In human tissue specimens, AAT-antigen blockade using anti-AAT antibody abolished ATZ11 staining of Z-AAT in a heterozygous AAT-deficient patient, whereas VWF-antigen blockade using anti-VWF abolished ATZ11 staining of endothelial cells and megakaryocytes.ConclusionsATZ11 reacts with cellular bound and denatured rVWF-WT and human VWF as shown using immunocytochemistry and subsequent confocal imaging, immunoelectron microscopy, SDS-PAGE and WB, and immunohistology. These immunoreactions are independent of the binding of Z-AAT-molecules and non-Z-AAT complexes.

研究目的
ATZ11抗体已被广泛用于鉴定血液样本与肝组织中的α1-抗胰蛋白酶（α1-anti-trypsin，AAT）PiZ型（Z-AAT）分子。本研究系统分析了该抗体在人体组织中的额外结合位点。

方法与结果
采用免疫电镜技术，探究ATZ11抗体在人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVECs）及健康个体血小板中的超微结构结合特征。将人胚肾293（HEK293）细胞用血管性血友病因子（Von Willebrand factor，VWF）进行瞬时转染，通过免疫细胞化学法结合共聚焦显微镜、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）及免疫印迹（WB）开展分析。对血管性血友病因子正常及缺乏患者的血小板与血清样本，采用非变性PAGE、SDS-PAGE联合WB进行检测。通过针对AAT与VWF抗原的大规模抗体阻断实验，从免疫组织化学层面验证ATZ11反应的特异性。
在HUVEC的韦贝尔-帕拉德小体（Weibel-Palade bodies，WPBs）及血小板α颗粒中，可检测到ATZ11阳性表位。ATZ11可对HEK293细胞中携带重组野生型VWF（rVWF-WT）的假WPB进行染色。在SDS-PAGE联合WB实验中，抗VWF抗体与ATZ11均可识别rVWF-WT。但通过WB检测时，ATZ11无法识别rVWF-WT多聚体、人VWF多聚体以及VWF缺乏患者的血清蛋白；而抗VWF抗体（anti-VWF）则可同时识别rVWF-WT多聚体与人VWF多聚体。在人体组织标本中，针对杂合子α1-抗胰蛋白酶缺乏症患者，采用抗AAT抗体阻断AAT抗原后，ATZ11对Z-AAT的染色信号消失；而采用抗VWF抗体阻断VWF抗原后，ATZ11对内皮细胞与巨核细胞的染色信号消失。

结论
通过免疫细胞化学与共聚焦成像、免疫电镜、SDS-PAGE及WB、免疫组织化学实验证实，ATZ11可与细胞结合型及变性的重组野生型VWF与人VWF发生反应。此类免疫反应不依赖于Z-AAT分子与非Z-AAT复合物的结合。