Quantification of Shiga Toxins in Bacterial Cells

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).