From Computationally Aided Hapten Design to Fluorescent Biosensing: A Novel Strategy for Highly Sensitive Simultaneous Detection of Amatoxins and Phallotoxins in Mushrooms

Lethal amatoxins (AMAs) and toxic phallotoxins (PHLs) are the primary causes of mushroom poisoning, necessitating rapid detection methods. Utilizing similarity and quantum chemical analyses for hapten screening, this study successfully developed monoclonal antibody (mAb) 3A9 with high and uniform sensitivity to phalloidin and phallacidin (IC₅₀: 1.32 and 1.52 ng/mL). Furthermore, the heterologous hapten α-AMA-HS greatly improved the uniform recognition of mAb 3G9 to α-, β-, and γ-AMA (IC₅₀: 0.46, 0.67, and 0.51 ng/mL). Using these mAbs, a dual-target fluorescent immunochromatographic assay (DT-FICA) was developed to detect PHLs and AMAs simultaneously in mushrooms. It showed calculated limits of detection of 3.28 and 1.24 μg/kg in dry weight and 1.08 and 1.00 μg/kg in fresh weight. DT-FICA has demonstrated its accuracy and reliability through spiked recovery tests and real-sample analysis, providing a sensitive, specific, simple, and cost-effective tool for preventing mushroom poisoning and safeguarding public health.