Alkali-insoluble and soluble fungal cell wall fractions were obtained as described previously34 (link). For the ELISA, wells were coated overnight with 20-200 µg/mL of the cell wall fractions, melanin ghosts (in house generated), purified YWA1 (in house generated), 1,8-DHN, 1,2-DHN, 1,4-DHN, naphthalene, 1-naphthol (all from Sigma) in 50 mM carbonate buffer pH 9.6, and then blocked with 1% BSA in PBS. Fc-MelLec (5 µg/mL) was added to the wells and incubated for 1 h at room temperature. Following washing with PBS-Tween-20 (0.5% (v/v)), peroxidase-conjugated human Fc-specific IgG (Sigma) was added to the wells and incubated for 1 h at room temperature. Following further washing, quantification of Fc-MelLec binding was detected using ortho-phenylenediamine (OPD, Sigma) and H2O2 detection system (Merck). The reaction was stopped using 4% (v/v) H2SO4 and optical densities measured at 492 nm.
Protocol detail
Characterization of Fungal Cell Wall Interactions
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1,8-DHN
1,2-DHN
1,4-DHN
naphthalene
1-naphthol
peroxidase-conjugated human Fc-specific IgG
H2O2 detection system
1 naphthol
Alkali
Buffer
Carbonate
Cell wall
Elisa
Ghosts
H2o2
Human
Melanin
Naphthalene
Optical
Ortho phenylenediamine
Peroxidase
Tween 20
Corresponding Organization :
Other organizations : University of Aberdeen, Medical Research Council, Institut Pasteur, University of Minho, Imperial College London, Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie e. V. - Hans-Knöll-Institut (HKI), Friedrich Schiller University Jena, National Institutes of Health, National Institute of Allergy and Infectious Diseases, Radboud University Nijmegen, Radboud University Medical Center, University of Lisbon, IPO Porto
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Variable analysis
independent variables
- Cell wall fractions (alkali-insoluble and soluble fungal)
- Melanin ghosts (in-house generated)
- Purified YWA1 (in-house generated)
- 1,8-DHN
- 1,2-DHN
- 1,4-DHN
- Naphthalene
- 1-naphthol
- Fc-MelLec binding
- Coating concentration (20-200 μg/mL)
- Carbonate buffer pH 9.6
- Blocking with 1% BSA in PBS
- Fc-MelLec concentration (5 μg/mL)
- Incubation time (1 hour at room temperature)
- PBS-Tween-20 (0.5% (v/v)) for washing
- Peroxidase-conjugated human Fc-specific IgG
- OPD and H2O2 detection system
- 4% (v/v) H2SO4 to stop the reaction
- Optical density measurement at 492 nm
- Melanin ghosts (in-house generated)
- Purified YWA1 (in-house generated)
- 1,8-DHN
- 1,2-DHN
- 1,4-DHN
- Naphthalene
- 1-naphthol
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