Recombinant antigens as diagnostic markers for aspergillosis

Sarfati, J.; Monod, M.; Recco, P.; Sulahian, Annie; Pinel, Claudine; Candolfi, Ermanno; Fontaine, Thierry; Debeaupuis, J P; Tabouret, Marc; Latgé, Jean‐Paul

doi:10.1016/j.diagmicrobio.2006.02.002

Cited by 92 publications

(121 citation statements)

References 48 publications

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“…Significance of the regression was estimated with the t-test and r 2 values were computed according to the JMP software. OD values obtained for DPPV and CAT antigens were obtained in a previous study (Sarfati et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

“…The GPI proteins are produced in soluble form in Pichia pastoris, which is known to date as the most efficient heterologous host to produce recombinant proteins, particularly of A. fumigatus. High amounts of recombinant proteins (5-50 µg/ml) are produced extracellularly and are easily purified on a Ni column after a 6 × His tagging of the protein (Sarfati et al, 2006). We found that both patient populations are able to mount an antibody response against recombinant proteins of the Gel/Gas and Crh/Crf families.…”

Section: -Glucan Inmentioning

confidence: 98%

“…After binding and washing the column with a 20 mM phosphate buffer, pH 6, containing 0.5 M NaCl, proteins were eluted from the Ni 2+ column with 50 mM histidine solution in water. The solubilization of the different antigens in the 50 mM histidine solution allowed a direct coating of the antigens on the microtitre plate, whereas imidazole interfered with the coating and subsequent colorimetric reactions (Sarfati et al, 2006).…”

Section: Production Of Recombinant Polypeptidesmentioning

confidence: 99%

“…These patients have been classified previously and used for antibody detection in another study (Sarfati et al, 2006); all positive patients displayed 4-20 precipitin bands on immunoelectrophoresis. Candida patients (37) were from the Hôpital Cochin (Paris, France) and were classified based on clinical signs and antibody detection.…”

Section: Human Seramentioning

confidence: 99%

“…After drying, the plates were stored frozen. Patient sera were diluted 1/500 in PBS containing 0.05% Tween 20 and 1% BSA, and antibody measurement was performed as previously described (Sarfati et al, 2006), using as a secondary antibody an anti IgG (H + L) antibody conjugated to peroxidase. Detection was performed using orthophenylene diamine and the absorbance measured at 490 nm.…”

Section: Antigen Coating and Elisamentioning

confidence: 99%

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The GPI‐anchored Gas and Crh families are fungal antigens

Arroyo

Sarfati

Baixench

et al. 2007

Yeast

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The cell wall is the first interface between a fungus and its extracellular environment. Glycosyltransferases involved in the formation and dynamic remodelling of the polysaccharide network of the cell wall have recently been identified. The best characterized ones belong to the Gas family, which elongates β(1,3)-glucans, and to the Crh family, which are involved in the cross-linking of chitin to β(1,6)-glucan. All these proteins carry a glycosylphosphatidylinositol (GPI) anchor. In this work, we show that recombinant soluble forms of Gas1-5 and Crh1p from Saccharomyces cerevisiae and their orthologous proteins Gel1-Gel2 and Crf1 from Aspergillus fumigatus are specifically recognized by antibodies present in the sera of patients with Aspergillus or Candida infections. Quantification of the antibody titres against recombinant Gas/Gel and Crh/Crf proteins separated aspergilloma and candidiasis patients from non-infected individuals. Cross-reactivity was seen between the antibody response of patients with aspergillosis and candidiasis towards the Gas/Gel and Crh/Crf proteins. These results suggest that GPI-anchored cross-linking enzymes are relevant immunologically reactive constituents of the cell wall that may play a role during human fungal infections.

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Section: Discussionmentioning

confidence: 99%

Section: -Glucan Inmentioning

confidence: 98%

Section: Production Of Recombinant Polypeptidesmentioning

confidence: 99%

Section: Human Seramentioning

confidence: 99%

Section: Antigen Coating and Elisamentioning

confidence: 99%

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The GPI‐anchored Gas and Crh families are fungal antigens

Arroyo

Sarfati

Baixench

et al. 2007

Yeast

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Development of an HPTLC‐based diagnostic method for invasive aspergillosis

Puri

Ahmad

Panda

2010

Biomedical Chromatography

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A rapid, sensitive and specific high-performance thin-layer chromatographic (HPTLC) method was developed and validated for determination of gliotoxin in Aspergillus infected immunocompromised patients with invasive aspergillosis (IA). Densitometric analysis of gliotoxin was carried out in the absorbance mode at 254 nm after single-step extraction with chloroform. The method uses TLC aluminum plates pre-coated with silica gel 60F-254 as a stationary phase and toluene-isoamyl alcohol-methanol (10:0.5:0.5, v/v/v) as mobile phase, which gives compact spot of gliotoxin (R(f) = 0.51). The calibration curve was linear (r(2) > or = 0.994) between peak area and concentration in the tested range of 100-1000 ng spot(-1) with minimum detectable range 0.025 ng mu(-1) of serum sample. The mean +/- SD value of slope and intercept of the standard chromatogram of gliotoxin were found to be 523.2 +/- 1.555635 and 915.8 +/- 30.68843, respectively. The developed method is simple, rapid, precise and less costly than earlier diagnostic methods, and different serum samples can be run on a single TLC plate for comparative analysis. The proposed method can be used to analyze gliotoxin in patient serum for easy, rapid and cost-effective diagnosis of IA.

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Synthesis of a Pentasaccharide and Neoglycoconjugates Related to Fungal α‐(1→3)‐Glucan and Their Use in the Generation of Antibodies to Trace Aspergillus fumigatus Cell Wall

Komarova

Orekhova

Tsvetkov

et al. 2014

Chemistry A European J

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3-Aminopropyl α-(1→3)-pentaglucoside, a fragment of α-(1→3)-glucan of the cell wall of Aspergillus fumigatus, has been synthesized in a blockwise approach. The application of mono- and disaccharide N-phenyltrifluoroacetimidates bearing a stereodirecting 6-O-benzoyl group was essential for stereoselective α-glucosylations. In the products, p-methoxyphenyl and levulinoyl groups served as orthogonal protecting groups for the anomeric position and 3-OH group, respectively. Their removal from shared blocks led to donors and acceptors that were used for the synthesis of pentasaccharides. Coupling of free α-(1→3)-pentaglucoside with biotin and bovine serum albumin (BSA) gave glycoconjugate tools for mycological studies. Immunization of mice with the BSA conjugate induced the generation of antibodies that recognize α-(1→3)-glucan on A. fumigatus cell wall and distinguish its morphotypes. This discovery represents a first step to the development of a diagnostic test system and a vaccine to detect and fight this life-threatening pathogen.

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Recombinant antigens as diagnostic markers for aspergillosis

Cited by 92 publications

References 48 publications

The GPI‐anchored Gas and Crh families are fungal antigens

The GPI‐anchored Gas and Crh families are fungal antigens

Development of an HPTLC‐based diagnostic method for invasive aspergillosis

Synthesis of a Pentasaccharide and Neoglycoconjugates Related to Fungal α‐(1→3)‐Glucan and Their Use in the Generation of Antibodies to Trace Aspergillus fumigatus Cell Wall

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