A robust method for quantitative detection of β-1,3-glucan using Limulus polyphemus factor Gα fragment

β-1,3-glucan is an important glycan in the cell wall of most fungi and plants. Accurate detection of β-1,3-glucans is essential for their use in the food and pharmaceutical industries. Here, we developed a robust method to detect β-1,3-glucan using Limulus polyphemus factor Gα (LFGα) fragments. Five β-1,3-glucans with various degrees of branching were extracted from various edible mushrooms and structurally characterized. Their binding affinities to truncated LFGα variants (Q + D1 + D2, D1 + D2, D1, D2) were evaluated using the ELISA assay. LFGα fragment Q + D1 + D2 exhibited the highest affinity toward all β-1,3-glucans regardless of the degree of branching, whereas fragments D1 + D2, D1, and D2 had reduced affinities as the degree of branching increased. AlphaFold-predicted models and molecular docking supported these findings. The branching-insensitive binding with Q + D1 + D2 enabled us to accurately quantify the amount of β-1,3-glucan using a Q + D1 + D2-based ELISA assay. A standard curve was established using commercial curdlan, and quantitative detection of five mushroom β-1,3-glucans was in agreement with their actual glucan content. Our results demonstrate that the Q + D1 + D2-based ELISA assay can be developed as an efficient method for quantitative detection of β-1,3-glucans.