Ridascreen verotoxin microtiter plates

Sandwich-ELISA was used to detect Stx2 in the bacterial culture supernatants, and was carried out in RIDASCREEN Verotoxin microtiter plates (R-Biopharm GmbH, Darmstadt, Germany) coated with capture antibodies for detecting both Stx1 and Stx2. A monoclonal antibody against Stx2 (LSBio, WA, USA) conjugated with horseradish peroxidase using Peroxidase Labeling Kit–NH₂ (Dojindo, Kumamoto, Japan) was used as the detection antibody. A standard curve was determined with 2-fold serial dilutions of purified Stx2 (provided by Denka Seiken Co. Ltd.). Detection was carried out with urea peroxidase/TMB substrate reagent and 1 M sulfuric acid as the stop reagent, both were provided in the RIDASCREEN Verotoxin kit. Absorbance at 450 nm (A₄₅₀) was measured using a 96-well plate reader (Multiscan FC, Thermo Fisher Scientific, MA, USA).

We inoculated bacterial cells into 40 mL of CAYE broth (Merck, Darmstadt, Germany) and grew them to mid-log phase at 37°C with shaking. We then added mitomycin C (MMC) to the culture at a final concentration of 500 ng/mL. After MMC addition, we collected 100 μL of the culture every hour and immediately subjected it to sonication using a Bioruptor (Cosmo Bio, Tokyo, Japan). We obtained the soluble fractions of each cell lysate via centrifugation at 14,000 × g for 10 min at 4°C. We determined Stx1 and Stx2 concentrations in each cell lysate using a previously described sandwich ELISA (40 (link)). We captured Stx using RIDASCREEN Verotoxin microtiter plates (R-Biopharm, Darmstadt, Germany) coated with capture antibodies that recognize both Stx1 and Stx2. We conjugated monoclonal antibodies against Stx1 and Stx2 (LSBio, Seattle, WA, USA) with horseradish peroxidase using the Peroxidase Labeling Kit–NH₂ (Dojindo, Kumamoto, Japan) and employed them as detection antibodies. We used reagents supplied in the RIDASCREEN Verotoxin kit for detection. Finally, we measured the absorbance at 450 nm (A₄₅₀) using Tecan Infinite 200 PRO (Tecan, Männedorf, Switzerland).