Identification of a putative Crf splice variant and generation of recombinant antibodies for the specific detection of Aspergillus fumigatus

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...

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Bibliographic Details
Published in:PloS one 2009-08, Vol.4 (8), p.e6625-e6625
Main Authors: Schütte, Mark, Thullier, Philippe, Pelat, Thibaut, Wezler, Xenia, Rosenstock, Philip, Hinz, Dominik, Kirsch, Martina Inga, Hasenberg, Mike, Frank, Ronald, Schirrmann, Thomas, Gunzer, Matthias, Hust, Michael, Dübel, Stefan
Format: Article
Language:English
Subjects:
Alternative splicing
Amino Acid Sequence
Animals
Antibodies
Antibodies - immunology
Antifungal agents
Antigenic determinants
Antigens
Aspergillosis
Aspergillosis - diagnosis
Aspergillosis - microbiology
Aspergillus fumigatus
Aspergillus fumigatus - immunology
Aspergillus fumigatus - isolation & purification
Bacillus anthracis
Base Sequence
Biotechnology/Applied Microbiology
Biotechnology/Bioengineering
Candida albicans
Chemical synthesis
Cloning
Conformation
DNA, Complementary
Drugs
Enzyme-Linked Immunosorbent Assay
Epitope Mapping
Epitopes
Fungal Proteins - chemistry
Fungal Proteins - immunology
Fungi
Fungicides
Genomic libraries
Humans
Hyphae
Immunization
Immunocompromised hosts
Immunofluorescence
Immunology
Immunology/Immune Response
Immunosuppressive agents
Laboratories
Macaca
Medical screening
Membrane Proteins - genetics
Membrane Proteins - immunology
Mice
Microbiology/Immunity to Infections
Microbiology/Medical Microbiology
Microscopy
Molecular Sequence Data
Mycoses
Peptides
Phage display
Phages
Prognosis
Proteins
Recombinant Proteins - chemistry
Recombinant Proteins - immunology
RNA Splicing
Sequence Homology, Amino Acid
Sequence Homology, Nucleic Acid
Spores
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Summary: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. 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. Crf2 and the corresponding recombinant antibodies offer a novel approach for the early diagnostics of IA caused by A. fumigatus.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0006625