Quantification of Shiga Toxin 2

Bacterial cells were inoculated into 40 ml of lysogeny broth and grown to mid-log phase at 37 °C with shaking. MMC was then added to the culture at a final concentration of 500 ng/ml. At 5 h after MMC addition, 100 µl of the culture was collected and immediately subjected to sonication using a Bioruptor (Cosmo Bio, Tokyo, Japan). The soluble fractions of each cell lysate were obtained via centrifugation at 14,000 × g at 4 °C for 10 min. The Stx2 concentration in each cell lysate was determined using a previously described sandwich ELISA [69 (link)] using RIDASCREEN Verotoxin microtiter plates (R-Biopharm GmbH, Germany) coated with capture antibodies that recognize Stx2 and monoclonal antibodies against Stx2 (LSBio, WA, USA) conjugated with horseradish peroxidase using a Peroxidase Labeling Kit–NH2 (Dojindo, Kumamoto, Japan) as detection antibodies. Note that both Stx2a and Stx2g could be quantified at a certain level of accuracy using the detection antibodies [70 (link)].