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.ResultsThe 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.ConclusionCrf2 and the corresponding recombinant antibodies offer a novel approach for the early diagnostics of IA caused by A. fumigatus.

Identification of Immunogenic Antigens from Aspergillus fumigatus by Direct Multiparameter Characterization of Specific Conventional and Regulatory CD4+ T Cells

“…So far, CD4 + target proteins primarily have been identified indirectly via the presence of isotype-switched Abs (25,(42)(43)(44)(45)(46)(47). Alternatively, functional T cell assays, such as proliferation or cytokine production, have been used in a few studies (31-33, 38, 39, 48, 49) but do not allow for quantitative enumeration and phenotypic characterization of the reactive T cells.…”

“…The recombinant Crf2 (AFUA_1G16190) protein was generated as described (25). The recombinant GliT protein (AFUA_6G09740) was produced in E. coli and purified as described (26).…”

Generation and characterization of protective antibodies to Marburg virus

“…22,28,29 In brief, the secondary PCRs were carried out for each forward oligonucleotide primers separately to keep the diversity. Each PCR was performed in a volume of 100 ml using 100 ng purified PCR reaction product of the pGemT cloned cDNA, 2.5 U Go Taq polymerase (Promega, Mannheim, Germany), 200 mM dNTPs each and 200 nM of each oligonucleotide primer for 20 cycles (30 s 94 C, 30 s 57 C, 30 s 72 C), followed by 10 min 72 C. The PCR products were separated by 1.5% (w/v) agarose gel, cut out and purified using Nucleospin Extract II Kit (Macherey-Nagel, D€ uren, Germany) according to the manufacturer's instructions.…”

Biomarkers for Invasive Aspergillosis: The Challenges Continue

“…These include the surface protein Cf2, which was isolated from a human patient with proven IA and is also present in growing hypha of A. fumigatus but not in spores [107] as is Asp f6/MnSOD [108], gliotoxin, which is produced by most A. fumigatus strains and is present in plasma and serum [109] and its inactive derivative bis(methylthio)gliotoxin, which is also recoverable from blood [110], 15 immunogenic proteins of the immunosecretome [111] and secreted proteases whose activity in serum specimens might serve as a new diagnostic approach if used with reporter peptides [112].…”