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Development of a Highly Sensitive β-Glucan Detection System Using Scanning Single-Molecule Counting Method.

Yoshiyuki Adachi¹, Hidetaka Nakata², Tetsuya Tanabe²

¹Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

International Journal of Molecular Sciences

|July 2, 2021

Summary

A new diagnostic method using recombinant proteins and scanning single-molecule counting (SSMC) offers an alternative to the Limulus amebocyte lysate (LAL) assay for detecting fungal infections. This approach reduces false positives from plant-derived glucans, improving accuracy in diagnosing invasive fungal infections.

Keywords:

(1→6)-β-glucanase Limulus amebocyte lysate assay beta-glucan recognition protein invasive fungal infection scanning single-molecule counting method

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Area of Science:

Mycology
Biotechnology
Medical Diagnostics

Background:

The Limulus amebocyte lysate (LAL) assay is widely used for diagnosing invasive fungal infections but suffers from limitations, including cross-reactivity with plant-derived glucans, leading to false positives.
Accurate and reliable detection of fungal infections is crucial for timely and effective patient treatment.

Purpose of the Study:

To develop a novel diagnostic method for invasive fungal infections that overcomes the limitations of the LAL assay.
To evaluate a new detection system combining recombinant β-glucan binding proteins with scanning single-molecule counting (SSMC).

Main Methods:

Preparation of a novel (1→3)-β-D-glucan recognition protein (S-BGRP) and a (1→6)-β-glucanase mutant protein.
Testing the binding affinity of these recombinant proteins to fungal β-glucans from *Candida* and *Aspergillus* species.
Utilizing the scanning single-molecule counting (SSMC) method for sensitive detection of β-glucan levels.

Main Results:

The S-BGRP and mutant protein demonstrated specific binding to fungal β-glucans.
The SSMC method showed significantly reduced sensitivity to plant-derived (1→3)-β-D-glucan compared to the LAL assay.
This reduction in cross-reactivity minimizes false positives, a known issue with the LAL method.

Conclusions:

The developed detection system using recombinant β-glucan-binding proteins and SSMC is a promising alternative to the LAL assay for diagnosing fungal infections.
This novel method offers improved specificity by reducing false positives associated with plant-derived glucans.
The SSMC-based system represents a significant advancement in the accurate and reliable diagnosis of invasive fungal infections.