Glass culture tubes

Acetonitrile and methanol were reagent grade or better and were purchased from Carlo Erba Reagents (Milan, Italy). Ultrapure water was produced by a Milli-Q^® Direct system (Merck KGaA, Darmstadt, Germany). T-2 and HT-2, sodium chloride (NaCl), sodium azide (NaN₃), phosphate buffer saline (PBS) and ovalbumin (OVA) were purchased from Sigma-Aldrich (Milan, Italy). Ten monoclonal antibodies (MAbs) specific for T-2G (clones 1–2, 1–3, 1–4, 2–5, 2–11, 2–13, 2–16, 2–17, 2–21 and 2–44) were produced by US Department of Agriculture-Agricultural Research Center (USDA-ARS, Peoria, IL, USA) and have been described in Maragos et al. [29 (link)]. A Mab specific for T-2 (clone 1) was produced by Chinese Academy of Agricultural Sciences—Oil Crops Research Institute (Wuhan, China) and has been described by Zhang et al. [35 (link)]. A Mab specific for HT-2 (clone H10-A10) was purchased from University of Natural Resources and Life Science of Vienna—Department for Agrobiotechnology IFA-Tulln (Tulln, Austria). T-2 glucoside and HT-2 glucoside, both as α-anomers, were produced by USDA-ARS and have been described by McCormick et al. [36 (link)]. Glass culture tubes (10 × 75 mm) were obtained from VWR International (Milan, Italy). Paper filters (No. 4) and glass microfiber filters (GF/A) were purchased from Whatman (Maidstone, UK).

To extract intracellular cyanopeptides, aliquots of Planktothrix cells after 14 or 28 days were harvested onto glass microfibre filters (pre-weighed and dried overnight at 40 °C; Whatman, GF/A, diameter 47 mm, ~1.6 µm) with a Millipore filtration apparatus. The glass microfibre filters with the harvested Planktothrix cells were dried overnight (40 °C) and were subsequently transferred to glass culture tubes (16 × 100 mm, VWR) with 14 mL of 80% aqueous methanol. Each sample test tube was vortexed and sonicated three times for 30 s intervals. Sample tubes were stored at −80 °C for 40 min and were subsequently allowed to thaw at room temperature. This freeze–thaw cycle, in combination with agitation and sonication, was repeated two additional times to induce cell lysis. Resulting intracellular cyanopeptides in the methanolic extracts were filtered through 0.22 µm PTFE syringe filters (25 mm; ChromSpec, Inc., Brockville, ON, Canada) into amber vials and dried under a gentle stream of nitrogen gas. The dried extracts were reconstituted in 1.5 mL of HPLC-grade methanol and transferred to 2 mL amber HPLC vials (Agilent, Santa Clara, CA, USA), dried under a nitrogen gas and stored at −20 °C until instrumental analysis. Data for the extraction of Planktothrix intracellular compounds are reported in Supplementary Materials Table S2.

The concentrations of OTA standard working solution were 0, 0.0034, 0.01, 0.03, 0.1, 0.3, 1, 3, and 9 ng/mL prepared with 10% methanol in deionized water. The FPIA was prepared by 0.1 M borate buffer (pH = 7.4); the antibody working fluid was based on attenuating OTA specific antibody (mAb) 1:36,000 in BB buffer. Glass culture tubes with specifications 10 × 75 mm (VWR Scientific) were used as test cuvettes. We added 500 µL antibody working solution into the tube, then 500 µL OTA-EDF (or OTA-BDF/OTA-AMF/OTA-HDF) working solution and mixed. The FP value was measured after 10 min of oscillate incubation at ambient temperature; the relative FP mean values (mP/mP₀) were used in the inhibition curve to standardize the FP value, where mP is the current FP value of different OTA concentrations and mP₀ is the value of blank-control (50 µL methanol-BB solution was used as the blank-control) [41 (link)]. The values of mP/mP₀ were plotted against OTA concentration [37 (link)]. For experiments to elucidate the reaction’s kinetics, measurements were recorded for time intervals ranging from 3 s to 10 min at room temperature, unless otherwise noted. OTA content of naturally contaminated rice samples was approximated based on the OTA-PBS solution specification curve [42 (link)].