Ridascreen verotoxin kit

Shiga toxins produced by E. coli O157:H7 EDL 933 in the ileal effluents of the TIM system were dosed by enzyme linked immunosorbent assay (ELISA) using the Ridascreen^®Verotoxin kit (R-Biopharm, Darmstadt, Germany). Purified toxin Stx2 (Toxin Technology^®, Sarasota, FL, USA) was used to establish standard calibration curves. Samples were diluted to half with the provided diluent and the dosage was made according to the manufacturer’s instructions. Each digestive sample was analyzed in duplicate and serial dilutions of purified toxin were analyzed in triplicate.

A volume of 100 µL overnight LB-cultures was transferred to 5 mL fresh LB and incubated at 37 °C with agitation at 250 rpm until the optical density reached 0.5 at 600 nm (OD₆₀₀). Half of these cultures were induced by addition of 0.5 µg/mL of Mitomycin C (MMC). Both induced and uninduced cultures were incubated further for 3 h. Six samples, three induced and thee uninduced, were processed and analyzed with respect to Stx content for each STEC isolate. The Stx content was measured in 1:20 dilutions of the cultures using the semi-quantitative enzyme immunoassay RIDASCREEN^® Verotoxin kit (R-biopharm, Darmstadt, Germany) according to the manufacturer.