Abstract:Based on the principle of enzyme-linked immunosorbent assay（ELISA）， an ELISA kit for detecting amanitin in mushrooms was developed. Using N-hydroxysuccinimide （NHS） and 1-（3-dimethylaminopropyl）-3-ethylcarbodiimide （EDC） as activators， a semi-antigen of amanitin was prepared and then coupled with keyhole limpet hemocyanin （KLH） and bovine serum albumin （BSA） to prepare immunogens and coating antigens. The immunogens were used to immunize Balb/c mice， which were then used to produce monoclonal antibodies. Through optimization of the optimal antigen coating concentration and the optimal dilution ratio of enzyme-linked antibodies， an ELISA kit for amanitin was successfully developed. The sensitivity， accuracy， and precision of the kit were evaluated. The linear range of the reagent kit was 0.1-3.6 ng/mL， with a half-maximal inhibitory concentration （IC50） of 0.5 ng/mL. The cross-reactivity of the kit for α-amanitin and γ-amanitin was 32% and 38%， respectively， while the cross-reactivity with the structurally similar phalloidin was less than 0.50%， demonstrating good specificity of the kit. The recovery rate for added β-amanitin was between 101.0%and 106.0%， with an intra-run coefficient of variation （CV） of ≤5.22% and an inter-run CV of ≤9.24%. This is reagent kit for on-site screening and rapid detection of amanita toxin peptides in mushrooms.