Development of a Vertical Flow Dot-Immunobinding Assay (Dot-Iba) for Rapid Detection of Neoscytalidium dimidiatum

Abstract

Neoscytalidium dimidiatum is a non-dermatophyte mold that commonly causes skin and nail infections in tropical regions and often resists conventional antifungal therapies. Because its clinical and laboratory features often resemble dermatophyte infections, diagnosis is frequently delayed and treatment is sometimes inappropriate. We therefore developed a dot-immunobinding assay (Dot-Iba) to detect N. dimidiatum antigens. We generated a highly specific monoclonal antibody, 3E6F7 (MAb 3E6F7), for antigen capture, and used goat anti-mouse Ig conjugated with alkaline phosphatase (AP) as the signal generator. The test pad comprised a test hole, a nitrocellulose membrane (NC), and water-absorbent pads in a vertical flow-through format to allow a rapid antigen-antibody reaction. The assembled system detected N. dimidiatum antigens in vitro with high specificity and yielded visible results within 2 h; its detection limit was 0.9 µg without cross-reactivity to dermatophyte or non-dermatophyte fungi. This rapid, specific, and easy-to-use assay shows strong potential as a diagnostic tool, particularly in settings with limited access to fungal culture or advanced molecular diagnostics, where early, accurate identification is crucial.