title: ATZ11 recognizes not only Z-α1-antitrypsin-polymers and complexed forms of non-Z-α1-antitrypsin but also the von Willebrand factor.
abstract: OBJECTIVE
  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.
  
  
  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.
authors.affiliation: Department of Pathology, University Bonn, Bonn, Germany.
authors.forename: Diane
authors.initials: D
authors.lastname: Goltz
authors.affiliation: Department of Pathology, University Bonn, Bonn, Germany.
authors.forename: Kanishka
authors.initials: K
authors.lastname: Hittetiya
authors.affiliation: Institute for Experimental Hematology, University Bonn, Bonn, Germany.
authors.forename: Hamideh
authors.initials: H
authors.lastname: Yadegari
authors.affiliation: Institute for Experimental Hematology, University Bonn, Bonn, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Driesen
authors.affiliation: Department of Pathology, University Bonn, Bonn, Germany.
authors.forename: Jutta
authors.initials: J
authors.lastname: Kirfel
authors.affiliation: Medical Clinic, St. Vincenz-Hospital, Limburg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Neuhaus
authors.affiliation: Department of Pathology, University Bonn, Bonn, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Steiner
authors.affiliation: Department of Pathology, University Bonn, Bonn, Germany.
authors.forename: Christiane
authors.initials: C
authors.lastname: Esch
authors.affiliation: Department of Pathology, University Bonn, Bonn, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Bedorf
authors.affiliation: Institute for Experimental Hematology, University Bonn, Bonn, Germany.
authors.forename: Hans-Jörg
authors.initials: HJ
authors.lastname: Hertfelder
authors.affiliation: Department of Pathology, University Bonn, Bonn, Germany.
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Fischer
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D001792:Blood Platelets / Q000737:chemistry / Q000276:immunology; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D007150:Immunohistochemistry; D016253:Microscopy, Immunoelectron; D011485:Protein Binding; D014162:Transfection; D020302:Weibel-Palade Bodies / Q000737:chemistry / Q000276:immunology; D000515:alpha 1-Antitrypsin / Q000737:chemistry* / Q000276:immunology; D014841:von Willebrand Factor / Q000737:chemistry* / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D000515:alpha 1-Antitrypsin; D014841:von Willebrand Factor