title: Identification of a putative Crf splice variant and generation of recombinant antibodies for the specific detection of Aspergillus fumigatus.
abstract: BACKGROUND
  Aspergillus fumigatus is a common airborne fungal pathogen for humans. It frequently causes an invasive aspergillosis (IA) in immunocompromised patients with poor prognosis. Potent antifungal drugs are very expensive and cause serious adverse effects. Their correct application requires an early and specific diagnosis of IA, which is still not properly achievable. This work aims to a specific detection of A. fumigatus by immunofluorescence and the generation of recombinant antibodies for the detection of A. fumigatus by ELISA.
  
  
  RESULTS
  The A. fumigatus antigen Crf2 was isolated from a human patient with proven IA. It is a novel variant of a group of surface proteins (Crf1, Asp f9, Asp f16) which belong to the glycosylhydrolase family. Single chain fragment variables (scFvs) were obtained by phage display from a human naive antibody gene library and an immune antibody gene library generated from a macaque immunized with recombinant Crf2. Two different selection strategies were performed and shown to influence the selection of scFvs recognizing the Crf2 antigen in its native conformation. Using these antibodies, Crf2 was localized in growing hyphae of A. fumigatus but not in spores. In addition, the antibodies allowed differentiation between A. fumigatus and related Aspergillus species or Candida albicans by immunofluorescence microscopy. The scFv antibody clones were further characterized for their affinity, the nature of their epitope, their serum stability and their detection limit of Crf2 in human serum.
  
  
  CONCLUSIONS
  Crf2 and the corresponding recombinant antibodies offer a novel approach for the early diagnostics of IA caused by A. fumigatus.
authors.affiliation: Technische Universität Braunschweig, Institut für Biochemie und Biotechnologie, Braunschweig, Germany.
authors.forename: Mark
authors.initials: M
authors.lastname: Schütte
authors.forename: Philippe
authors.initials: P
authors.lastname: Thullier
authors.forename: Thibaut
authors.initials: T
authors.lastname: Pelat
authors.forename: Xenia
authors.initials: X
authors.lastname: Wezler
authors.forename: Philip
authors.initials: P
authors.lastname: Rosenstock
authors.forename: Dominik
authors.initials: D
authors.lastname: Hinz
authors.forename: Martina Inga
authors.initials: MI
authors.lastname: Kirsch
authors.forename: Mike
authors.initials: M
authors.lastname: Hasenberg
authors.forename: Ronald
authors.initials: R
authors.lastname: Frank
authors.forename: Thomas
authors.initials: T
authors.lastname: Schirrmann
authors.forename: Matthias
authors.initials: M
authors.lastname: Gunzer
authors.forename: Michael
authors.initials: M
authors.lastname: Hust
authors.forename: Stefan
authors.initials: S
authors.lastname: Dübel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D001228:Aspergillosis / Q000175:diagnosis* / Q000382:microbiology; D001232:Aspergillus fumigatus / Q000276:immunology / Q000302:isolation & purification*; D001483:Base Sequence; D018076:DNA, Complementary; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D005656:Fungal Proteins / Q000737:chemistry / Q000276:immunology; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D012326:RNA Splicing; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
chemical_list: D000906:Antibodies; D018076:DNA, Complementary; D005656:Fungal Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins
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