Phallacidin

Indirect cELISAs were completed using a panel of inhibitors to determine the selectivity of the mAbs. The cELISA procedure was nearly the same as that described for the serum screening, except for the addition of inhibitors (50 µL), which were mixed with 50 µL of the antibody solution during the primary antibody incubation step. The inhibitors tested were α-AMA (≥90%, Enzo Life Sciences, Farmingdale, NY, USA), β-AMA (≥90%, Enzo), γ-AMA (≥90%, Enzo), microcystin-LR (≥95%, Enzo), nodularin (≥95%, Enzo), phalloidin (>90%, Enzo), phallacidin (≥85%, Sigma), pysilocybin (>99%, Cerilliant, Round Rock, TX, USA), muscimol (>99%, Abcam, Cambridge, MA, USA), ibotenic acid (>98%, Abcam). Each analyte stock was dissolved in dH₂O, then serially diluted into TBST, starting at the highest concentration of ,000 ng mL⁻¹, and assessed in triplicate. Data were analyzed using a 4-parameter logistic equation (GraphPad Prism 7 Software, La Jolla, CA, USA) to determine the concentration of inhibition at half of the maximal signal (IC₅₀). Cross-reactivity (%) was calculated as follows: (IC₅₀ α-AMA)/(IC₅₀ test inhibitor) × 100.

The toxins, α-AMA (≥90%), β-AMA (≥90%), γ-AMA (≥90%), microcystin-LR (≥95%), nodularin (≥95%), and phalloidin (>90%), were purchased from Enzo Life Sciences (Farmingdale, NY, USA). Phallacidin (≥85%), sodium periodate (NaIO₄, >99.8%), sodium borohydride (NaBH₄, 99%), dimethyl sulfoxide (DMSO), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol, Tween-20, keyhole limpet hemocyanin (KLH), and SuperSignal West Pico PLUS chemiluminescent substrate were purchased from Fisher Scientific (Waltham, MA, USA). Secondary antibody goat-anti-rabbit-horseradish peroxidase conjugate was purchased from Abcam (Cambridge, MA, USA). The hapten OSu-Glu-vc-PAB-C6-amanitin (LB) was purchased from Levena Biopharma (San Diego, CA, USA).

lyophilized Amanita exitialis (100 g) was provided by Professor Chen (College of Life Sciences, Hunan Normal University, Changsha, China). The Amanita exitialis powder was stored in a refrigerator at 2–8 °C. α-amanitin, β-amanitin, γ-amanitin, phallacidin, HPLC-grade water, acetonitrile, and ammonium acetate were obtained from Sigma-Aldrich (St. Louis, MO, USA). Normal saline (0.9% NaCl), ethyl alcohol, formalin, and hematoxylin and eosin were obtained from Sangon Biotech (Shanghai, China).