title: Rapid enzyme-linked immunosorbent assay for the detection of antibodies against human neutrophil antigens -1a, -1b, and -1c.
abstract: BACKGROUND
  Several methods exist for the detection of neutrophil antibodies; most of them, however, require fresh neutrophils. In this study, an enzyme-linked immunosorbent assay (ELISA) using recombinant HNA-1 antigens (rHNAs) was developed to detect HNA-1a, -1b, and -1c alloantibodies in serum samples.
  
  
  METHODS
  Soluble rHNA-1a, -1b, and -1c were isolated from culture supernatant of transfected insect cells. Purified rHNA antigens were immobilized on microtiter wells using antibody against V5-Tag protein. Sera were added, and bound antibodies were detected by enzyme-labeled secondary antibodies. In parallel, monoclonal antibody-immobilized granulocyte antigen (MAIGA) was performed with two different monoclonal antibodies (MoAbs) against FcγRIIIb (3G8 and BW209).
  
  
  RESULTS
  Fifteen MAIGA-positive sera containing HNA-1a alloantibodies were tested in ELISA. Thirteen of 15 (86.7%) MAIGA-positive sera captured by MoAbs 3G8 and/or BW209 reacted specifically with rHNA-1a. Four (26.7%) HNA-1a sera showed additional reaction with rHNA-1c. When anti-HNA-1b alloantibodies were analyzed in ELISA, 13 of 15 (86.7%) showed specific positive reaction with rHNA-1b, and 12 of 15 (80.0%) cross-reacted with rHNA-1c. Two HNA-1c sera reacted specifically with rHNA-1c. Immunoprecipitation analysis of all ELISA-negative HNA-1a and -1b sera did not show any specific band indicating false-positive reaction of these sera in MAIGA assay.
  
  
  CONCLUSIONS
  These results suggested that rapid ELISA using recombinant neutrophil antigens may provide a valuable method for rapid screening of human alloantibodies against HNA-1a, -1b, and -1c in patients with neutropenia and in blood donors.
authors.affiliation: Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University, Giessen, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Werth
authors.forename: Behnaz
authors.initials: B
authors.lastname: Bayat
authors.forename: Yudy
authors.initials: Y
authors.lastname: Tjahjono
authors.forename: John
authors.initials: J
authors.lastname: Eskandar
authors.forename: Heike
authors.initials: H
authors.lastname: Berghöfer
authors.forename: Gregor
authors.initials: G
authors.lastname: Bein
authors.forename: Ulrich J
authors.initials: UJ
authors.lastname: Sachs
authors.forename: Sentot
authors.initials: S
authors.lastname: Santoso
mesh_terms: D000906:Antibodies / Q000032:analysis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007519:Isoantigens / Q000032:analysis
chemical_list: D000906:Antibodies; D007519:Isoantigens; C067704:neutrophil-specific antigen NA1, human; C104384:neutrophil-specific antigen SH