ATZ11 recognizes not only Z-α1-antitrypsin-polymers and complexed forms of non-Z-α1-antitrypsin but also the von Willebrand factor.
In: PLoS ONE, Jg. 9 (2014), Heft 3, S. e91538
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Zugriff:
AIMS: The 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 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 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. 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.
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ATZ11 recognizes not only Z-α1-antitrypsin-polymers and complexed forms of non-Z-α1-antitrypsin but also the von Willebrand factor.
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Autor/in / Beteiligte Person: | Goltz, Diane ; Hittetiya, Kanishka ; Yadegari, Hamideh ; Driesen, Julia ; Kirfel, Jutta ; Neuhaus, Thomas ; Steiner, Susanne ; Esch, Christiane ; Bedorf, Jörg ; Hertfelder, Hans-Jörg ; Fischer, Hans-Peter |
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Zeitschrift: | PLoS ONE, Jg. 9 (2014), Heft 3, S. e91538 |
Veröffentlichung: | Public Library of Science (PLoS), 2014 |
Medientyp: | academicJournal |
ISSN: | 1932-6203 (print) ; 0287-6469 (print) |
DOI: | 10.1371/journal.pone.0091538 |
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