Sandwich ELISA for Stx1-2 Detection

Sandwich ELISAs were performed as previously described [23 (link)]. Briefly, 96-well black NUNC Maxisorp flat bottom plates (Thermo Scientific, Waltham, MA, USA) were coated with 100 µL/well of 1 µg/mL of the capture antibody, Stx1-2 [24 (link)] and incubated at 4 °C overnight. After overnight incubation, the plates were washed two times with 0.02 M Tris buffered saline with 0.9 % NaCl, pH 7.4, and 0.05% Tween-20 (TBST), blocked in 5% non-fat dry milk (NFDM)-TBST for an hour at 37 °C, and then incubated with samples for 1 h at 37 °C. The plates were washed with TBST six times and incubated with 100 ng/mL of detection antibody, Stx1 pAb [23 (link)], in NFDM-TBST. Goat anti-rabbit HRP (Promega, Madison, WI, USA), 10 ng/mL in NFDM-TBST, was added as a secondary antibody after washing six times with TBST. Plates were developed with 100 µL SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA), and luminescence counts per second (CPS) were read on a Victor 3 plate reader (Perkin Elmer, Shelton, CT, USA) for 0.1 s. Each treatment was performed in triplicate and repeated twice on two different days.