Identification of a putative Crf splice variant and generation of recombinant antibodies for the specific detection of Aspergillus fumigatus.
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.
Schütte
Mark
M
Technische Universität Braunschweig, Institut für Biochemie und Biotechnologie, Braunschweig, Germany.
Thullier
Philippe
P
Pelat
Thibaut
T
Wezler
Xenia
X
Rosenstock
Philip
P
Hinz
Dominik
D
Kirsch
Martina Inga
MI
Hasenberg
Mike
M
Frank
Ronald
R
Schirrmann
Thomas
T
Gunzer
Matthias
M
Hust
Michael
M
Dübel
Stefan
S
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
D000906:Antibodies; D018076:DNA, Complementary; D005656:Fungal Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins
Proc Natl Acad Sci U S A. 1998 Jan 20;95(2):669-74
9435250
J Clin Microbiol. 2009 Jan;47(1):129-33
19005145
Nat Biotechnol. 2005 Sep;23(9):1105-16
16151404
Nature. 2005 Dec 22;438(7071):1151-6
16372009
EMBO J. 1994 Jul 15;13(14):3245-60
8045255
Pediatr Pulmonol. 2008 Dec;43(12):1249-51
19009619
Infect Dis Clin North Am. 2002 Dec;16(4):875-94, vi
12512185
J Immunol Methods. 2005 Apr;299(1-2):47-62
15914190
J Biol Chem. 2000 May 19;275(20):14882-9
10809732
Nature. 1991 Jan 24;349(6307):293-9
1987490
Antimicrob Agents Chemother. 2007 Aug;51(8):2758-64
17517846
Antimicrob Agents Chemother. 2005 Aug;49(8):3414-20
16048955
Chest. 1999 Feb;115(2):364-70
10027433
J Mol Biol. 2008 Dec 31;384(5):1400-7
18976662
Trends Microbiol. 2001 Aug;9(8):382-9
11514221
J Mol Biol. 2004 Jul 16;340(4):783-95
15223320
Comb Chem High Throughput Screen. 2003 Mar;6(2):147-54
12678710
Protein Eng. 2003 Oct;16(10):753-9
14600205
Nucleic Acids Res. 2005 Jan 1;33(Database issue):D671-4
15608286
Eur J Radiol. 2009 Jul;71(1):55-60
18468830
Yeast. 2007 Apr;24(4):289-96
17397107
Curr Opin Microbiol. 2002 Aug;5(4):379-85
12160856
CSH Protoc. 2006 Sep 01;2006(4):
22485929
Infect Immun. 2005 Aug;73(8):4704-13
16040983
Zentralbl Bakteriol A. 1980;247(4):530-6
6779440
Clin Infect Dis. 2008 Dec 15;47(12):1507-12
18990068
Curr Pharm Biotechnol. 2008 Dec;9(6):439-46
19075684
Trends Biotechnol. 2004 Jan;22(1):8-14
14690617
Eur Radiol. 1998;8(2):306-12
9477289
J Immunol Methods. 2002 Sep 1;267(1):13-26
12135797
Mol Immunol. 2000 Dec;37(17):1067-77
11399324
Clin Infect Dis. 1998 Apr;26(4):781-803; quiz 804-5
9564455
Lancet. 2003 Oct 4;362(9390):1142-51
14550704
J Clin Microbiol. 1999 Apr;37(4):1221-3
10074559
Infect Immun. 2006 Jun;74(6):3222-31
16714549
Allergy. 2002 Aug;57(8):723-8
12121192
J Infect Dis. 2006 Mar 1;193(5):741-7
16453271
Electrophoresis. 2001 Aug;22(13):2812-23
11545413
Int J Antimicrob Agents. 2006 Nov;28(5):450-3
17034993
PLoS Pathog. 2007 Feb;3(2):e13
17274685
Clin Exp Immunol. 2005 Feb;139(2):257-67
15654824
Int Arch Allergy Immunol. 2001 Jan-Mar;124(1-3):43-7
11306922
Drugs. 2007;67(11):1567-601
17661528
BMC Cancer. 2007 Jan 31;7:25
17266749
Clin Exp Allergy. 2001 May;31(5):761-70
11422136
Med Mycol. 2005 May;43 Suppl 1:S243-6
16110816
Microbes Infect. 2002 Nov;4(13):1281-90
12443892
Med Mycol. 2007 Feb;45(1):17-26
17325940
Drugs. 2008;68(14):1941-62
18778118
Clin Microbiol Rev. 1999 Apr;12(2):310-50
10194462
Nucleic Acids Res. 2004 Jan 1;32(Database issue):D401-5
14681443
Methods Mol Biol. 2007;408:243-55
18314587
Clin Drug Investig. 2008;28(8):509-21
18598097
Intern Med. 2000 Mar;39(3):191-2
10772117
J Postgrad Med. 2005;51 Suppl 1:S37-41
16519254
BMC Biotechnol. 2008 Sep 02;8:66
18764933
J Exp Med. 2005 Sep 5;202(5):597-606
16147975
Lancet Infect Dis. 2005 Oct;5(10):609-22
16183515
J Clin Microbiol. 2004 Jun;42(6):2733-41
15184460