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.
Highlights
Accepted: 29 May 2021As the number of immunocompromised patients increases, the number of opportunistic infections is increasing annually [1]
Since it has been reported that some immunoglobulin preparations for intravenous injection may contain Limulus amebocyte lysate (LAL)-detectable BGs, we investigated the reactivity of the scanning single-molecule counting (SSMC)
Six immunoglobulin preparations were assayed by LAL and SSMC methods; the BG concentrations are shown as Pachyman and CSBG equivalents (Table 2)
Summary
As the number of immunocompromised patients increases, the number of opportunistic infections is increasing annually [1]. The β-D-glucan (BG) test is a valuable diagnostic standard that covers a wide range of invasive fungal infections that have a poor prognosis [2]. A BG-binding protein, by genetic modification of the modification of We the created insect BGRP [13]. 1,6-β-glucan by replacing catalytic glutamic acid residue with gluglutamine to eliminate its hydrolase activity [15]. →3)-β-Dglucan andto(1→6)-β-D-glucan, the combination of S-BGRP and 16BGM will (1 enable the glucan and (1→6)-β-D-glucan, the combination of S-BGRP and 16BGM will enable the detection of fungal BGs with high specificity [17]. We developed a prototype detection of fungal BGs with high specificity [17]. BG reactive probe using S-BGRP and 16BGM and employed the scanning single-molecule. BG reactive probe using S-BGRP and 16BGM and employed the scanning single-molecule counting (SSMC) method for detection.
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