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Solubilization of yeast cell-wall beta-(1-->3)-D-glucan by sodium hypochlorite oxidation and dimethyl sulfoxide
N Ohno1, M Uchiyama, A Tsuzuki
1Tokyo University of Pharmacy and Life Science, School of Pharmacy, Japan.
Abstract:
The limulus test is a well-established method for the diagnosis of both Gram-negative sepsis and invasive fungal infection. To diagnose fungal infections, a beta-(1-->3)-D-glucan-specific chromogenic kit (Fungitec G test MK) has been developed and applied clinically. We are concentrating our main efforts on developing a better standard to improve the precision of this method. To this end, we have successfully developed a protocol to obtain a soluble Candida spp. beta-(1-->3)-D-glucan (CSBG) by sodium hypochlorite (NaClO) oxidation and subsequent dimethyl sulfoxide (Me2SO) extraction (yield of 9.6 +/- 4.1%) of acetone-dried whole-cell preparations. The beta-glucan fraction is free from the cell-wall mannan, gives a symmetrical peak by gel filtration, and is soluble in dilute NaOH. The product is composed mainly of beta-(1-->3)- and beta-(1-->6)-D-glucosidic linkages. The specific activity of the beta-glucan is comparable with pachyman when combined with the Fungitec G test as the standard glucan and reacted as low as 10(-11) g/mL.
Insights
Researchers developed a soluble Candida spp. beta-(1-->3)-D-glucan (CSBG) standard to enhance the precision of fungal infection diagnostics. This new standard shows comparable specific activity to existing methods, improving diagnostic accuracy for beta-D-glucan detection.
Area of Science:
- Biochemistry
- Mycology
- Medical Diagnostics
Background:
- The Limulus test is a standard diagnostic method for Gram-negative sepsis and invasive fungal infections.
- A beta-(1-->3)-D-glucan-specific chromogenic kit (Fungitec G test MK) is clinically used for fungal infection diagnosis.
- Improving the precision of fungal infection diagnostics requires a better beta-D-glucan standard.
Purpose of the Study:
- To develop a soluble beta-(1-->3)-D-glucan standard from Candida spp.
- To improve the precision of fungal infection diagnostics using the Fungitec G test MK.
Main Methods:
- Developed a protocol for obtaining soluble Candida spp. beta-(1-->3)-D-glucan (CSBG) via sodium hypochlorite (NaClO) oxidation and dimethyl sulfoxide (Me2SO) extraction.
- Acetone-dried whole-cell preparations were used, yielding 9.6 +/- 4.1% CSBG.
- Characterized the CSBG fraction for purity (free from mannan), solubility, and linkage composition (beta-(1-->3) and beta-(1-->6)).
Main Results:
- Successfully produced a soluble beta-glucan fraction (CSBG) from Candida spp.
- The CSBG fraction was free from cell-wall mannan and soluble in dilute NaOH.
- The specific activity of the CSBG was comparable to pachyman when used with the Fungitec G test, detecting concentrations as low as 10(-11) g/mL.
Conclusions:
- A novel, soluble Candida spp. beta-(1-->3)-D-glucan (CSBG) standard has been successfully developed.
- The developed CSBG standard is suitable for improving the precision of beta-D-glucan-based fungal infection diagnostics.
- This advancement offers a more accurate method for detecting invasive fungal infections.