ATZ11 Recognizes Not Only Z-alpha(1)-Antitrypsin-Polymers and Complexed Forms of Non-Z-alpha(1)-Antitrypsin but Also the von Willebrand Factor

作者：Goltz Diane; Hittetiya Kanishka; Yadegari Hamideh; Driesen Julia; Kirfel Jutta; Neuhaus Thomas; Steiner Susanne; Esch Christiane; Bedorf Joerg; Hertfelder Hans Joerg; Fischer Hans Peter^*

来源：PLos One, 2014, 9(3): e91538.

DOI：10.1371/journal.pone.0091538

Aims: The ATZ11 antibody has been well established for the identification of alpha(1)-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 Results: Ultrastructural 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 alpha-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.
Conclusions: ATZ11 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.

• 出版日期2014-3-19