title: Epitope mapping of antibodies directed against the human neutrophil alloantigen 3a.
abstract: BACKGROUND
  Severe transfusion-related acute lung injury is often caused by antibodies directed against the human neutrophil alloantigen (HNA)-3a. HNA-3a results from an amino acid exchange (Arg154Gln) in the first extracellular loop of the choline transporter-like protein 2 (CTL2). The characteristics of the binding domain(s) of HNA-3a antibodies are unknown.
  
  
  METHODS
  For epitope mapping, a library of 23 different HNA-3a (R(154)) and three HNA-3b (Q(154)) peptides covering different parts of the first extracellular loop of CTL2 (aa(55-231)) was synthesized in Escherichia coli and tested by Western blot with two HNA-3a alloantibody-containing plasma samples and by enzyme immunoassay (EIA) with different HNA-3a- (n = 21) and HNA-3b- (n = 1) positive plasma samples.
  
  
  RESULTS
  Despite promising Western blot results using highly reactive plasma samples, we found widely varying reactivities of different HNA-3a plasmas in the EIA, with only 11 of 21 HNA-3a antibodies binding to any of the tested HNA-3a peptides and with no peptide recognized by more than nine of the 21 antibodies. The HNA-3b plasma did not react with R(154) peptides in the EIA nor with R(154) or Q(154) peptides in Western blot experiments. Plasma reactivity profiles with the peptides did not correlate with those observed using granulocyte agglutination and granulocyte immunofluorescence tests.
  
  
  CONCLUSIONS
  Binding of HNA-3a alloantibodies depends on the conformation of the intact CTL2 protein and their binding sites may differ substantially. Peptide-based assays for detection of HNA-3a antibodies bear the risk to be insensitive and require systematic validation with a large panel of antibodies.
authors.affiliation: Institut für Immunologie und Transfusionsmedizin, Ernst-Moritz-Arndt-Universität Greifswald, Germany.
authors.forename: Tom
authors.initials: T
authors.lastname: Berthold
authors.forename: Jan
authors.initials: J
authors.lastname: Wesche
authors.forename: Kathleen
authors.initials: K
authors.lastname: Kuhnert
authors.forename: Birgitt
authors.initials: B
authors.lastname: Fürll
authors.forename: Holger
authors.initials: H
authors.lastname: Hippe
authors.forename: Jens
authors.initials: J
authors.lastname: Hoppen
authors.forename: Angelika
authors.initials: A
authors.lastname: Reil
authors.forename: Stefan
authors.initials: S
authors.lastname: Muschter
authors.forename: Jürgen
authors.initials: J
authors.lastname: Bux
authors.forename: Andreas
authors.initials: A
authors.lastname: Greinacher
mesh_terms: D001803:Blood Transfusion; D015153:Blotting, Western; D018604:Epitope Mapping; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D007518:Isoantibodies / Q000737:chemistry / Q000276:immunology*; D007519:Isoantigens / Q000737:chemistry / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry; D008875:Middle Aged; D010949:Plasma / Q000276:immunology*
chemical_list: C518134:HNA-3a antigen, human; D007518:Isoantibodies; D007519:Isoantigens; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; C484524:SLC44A2 protein, human