Development of an Immunoassay Platform Targeting β-1,3- and β-1,6-Glucans for Rapid Detection of Fungi

Fungal infections pose diagnostic challenges in both human and veterinary medicine, as traditional detection methods such as fungal culture are time-consuming, microscopy is operator-dependent, and molecular detection assays often require specialized instrumentation and trained personnel, which can limit their routine clinical application. This study developed a sandwich immunoassay to detect β-1,3- and β-1,6-glucans, two major components of the fungal cell wall, based on two catalytically inactive glucanase mutants, LamA_E175Q and Neg1_E321Q. The sandwich ELISA exhibited higher detection sensitivity than conventional ITS-based PCR for Saccharomyces cerevisiae and Candida albicans under the conditions of this study. Using pre-coated plates, the sample-processing and detection workflow can be completed in approximately 40 min. It effectively detected a wide range of fungal species, including yeasts (Saccharomyces cerevisiae, Candida albicans) and filamentous fungi such as dermatophytes and non-dermatophyte molds. In a preliminary clinical cohort, the assay identified β-glucan signals in all 21 samples confirmed positive for dermatophytes, while no signal was detected in 20 negative samples, suggesting potential clinical applicability. This dual-enzyme sandwich immunoassay provides a rapid and low-cost complementary tool for broad-spectrum fungal screening, which may help guide further confirmatory diagnostics and timely clinical decision-making.