*4.5. Method of Fluorescence Polarization Immunoassay*

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]. The values of mP/mP0 were plotted against OTA concentration [37]. 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].
