title: A novel enzyme-linked immunosorbent assay method for the detection of human neutrophil antigen-2a antibodies.
abstract: BACKGROUND
  Antibodies to human neutrophil antigen (HNA)-2a are responsible for a number of immune-mediated neutropenia disorders. Although several methods exist for the identification of anti-HNA-2a, all these methods have several limitations. In this study, a solid-phase enzyme-linked immunosorbent assay (ELISA) using recombinant HNA-2a antigen (rHNA-2a) allowing rapid detection of HNA-2a antibodies was developed.
  
  
  METHODS
  Soluble rHNA-2 was generated by transfection of insect cells with CD177 vector. Purified rHNA-2a was immobilized on microtiter wells coated with anti-CD177 and was applied to analyze 10 sera containing HNA-2a antibodies. For the evaluation of the ELISA method, results were compared with the standard assay, MAIGA (monoclonal antibody antigen capture assay) for detection of neutrophil antibodies.
  
  
  RESULTS
  The specificity of HNA-2a antibodies in all sera was confirmed by immunoblotting. Sera were then tested simultaneously in ELISA and MAIGA assays. Nine of 10 sera showed positive reactions in ELISA, whereas only 9 of 10 sera reacted in the standard MAIGA assay. All HNA-2a antibodies were detectable in MAIGA when diluted sera were applied. No reaction was observed with different sera containing neutrophil-reactive antibodies (6 anti-HNA-1a, 4 anti-HNA-1b, and 20 anti-HLA Class I and II) in ELISA. All HNA-2a antibodies were detectable in MAIGA when diluted sera were applied. Notably, sera containing anti-proteinase 3 (PR3) from patients with Wegener's granulomatosis reacted in MAIGA. In contrast, this antibody showed no reaction in ELISA with purified rHNA-2a.
  
  
  CONCLUSIONS
  These results demonstrated that ELISA with rHNA-2a provides a good method for detecting HNA-2a antibodies in human serum. This assay enables to exclude the presence of autoantibody against PR3 in patient's sera, which cannot be differentiated from anti HNA-2a with current serologic methods.
authors.affiliation: From the Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University, Giessen, Germany.
authors.forename: Behnaz
authors.initials: B
authors.lastname: Bayat
authors.forename: Silke
authors.initials: S
authors.lastname: Werth
authors.forename: Ulrich J H
authors.initials: UJ
authors.lastname: Sachs
authors.forename: Sentot
authors.initials: S
authors.lastname: Santoso
mesh_terms: D000906:Antibodies / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D058851:GPI-Linked Proteins; D006801:Humans; D007519:Isoantigens / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000276:immunology; D053146:Myeloblastin / Q000276:immunology; D009504:Neutrophils / Q000276:immunology; D011956:Receptors, Cell Surface / Q000276:immunology
chemical_list: D000906:Antibodies; C407242:CD177 protein, human; D058851:GPI-Linked Proteins; D007519:Isoantigens; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C067704:neutrophil-specific antigen NA1, human; C072753:neutrophil-specific antigen NA2; D053146:Myeloblastin