Sandwich and Direct ELISA for Toxin Detection

Sandwich ELISA’s were performed as previously described [54 (link)]. Briefly, 96-well black NUNC Maxisorp flat bottom plates (Thermo Scientific, Waltham, MA, USA) were coated with 100 µL/well of 1 g/mL of the capture antibody and incubated at 4 °C overnight. After overnight incubation the plates were washed two times in 0.02 M Tris buffered saline with 0.9% NaCl, pH 7.4, and 0.05% Tween-20 (TBST), blocked in 5% NFDM-TBST for an hour, and then incubated with samples in either PBS or LB at room temperature for 1 h. The plates were washed in TBST six times and incubated with the indicated detection antibody (100 ng/mL in NFDM-TBST). Goat anti-mouse HRP, or goat anti-rabbit HRP was added as a secondary antibody after washing six times in TBST. Plates were developed with 100 µL SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA) and luminescence was read on a Victor3 plate reader (Perkin Elmer, Waltham, MA, USA) for 0.1 s. The limit of detection (LOD) was defined as the lowest toxin concentration at which the average ELISA reading was three standard deviations above the negative control. For direct ELISA’s, the plates were directly coated with 100 µL/well of 10 ng/mL of Stx2k toxin, followed by blocking, washing, and incubation with rabbit antisera at the indicated dilution and then goat-anti Rabbit-HRP as described above.