Development of a Highly Sensitive β-Glucan Detection System Using Scanning Single-Molecule Counting Method |
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Authors: | Yoshiyuki Adachi Hidetaka Nakata Tetsuya Tanabe Daisuke Yamanaka Takashi Kanno Ken-ichi Ishibashi Naohito Ohno |
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Affiliation: | 1.Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan; (D.Y.); (T.K.); (N.O.);2.Biological Evaluation Technology, Olympus Corporation, 2-3 Kuboyama-cho, Hachioji, Tokyo 192-8512, Japan; (H.N.); (T.T.);3.Department of Host Defense and Responses, Kagawa Nutrition University, 3-9-21 Chiyoda, Sakado 350-0288, Japan; |
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Abstract: | To overcome the limitations of the Limulus amebocyte lysate (LAL) assay method for the diagnosis of invasive fungal infection, we applied a reaction system combining recombinant β-glucan binding proteins and a scanning single-molecule counting (SSMC) method. A novel (1→3)-β-D-glucan recognition protein (S-BGRP) and a (1→6)-β-glucanase mutant protein were prepared and tested for the binding of (1→6)-branched (1→3)-β-D-glucan from fungi. S-BGRP and (1→6)-β-glucanase mutant proteins reacted with β-glucan from Candida and Aspergillus spp. Although LAL cross-reacted with plant-derived β-glucans, the new detection system using the SSMC method showed low sensitivity to plant (1→3)-β-D-glucan, which significantly improved the appearance of false positives, a recognized problem with the LAL method. Measurement of β-glucan levels by the SSMC method using recombinant β-glucan-binding proteins may be useful for the diagnosis of fungal infections. This study shows that this detection system could be a new alternative diagnostic method to the LAL method. |
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Keywords: | (1→ 6)-β -glucanase beta-glucan recognition protein invasive fungal infection Limulus amebocyte lysate assay scanning single-molecule counting method |
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