Quantification of C. difficile Toxins

The three strains cultured in TY medium for 12, 24, 48, and 72 h were centrifuged to collect the supernatants containing TcdA and TcdB toxins; the supernatant was filtered with a 0.45 µm filter. Next, a 96-well plate was coated with the supernatant at 4 °C overnight and then washed with PBST (phosphate-buffered saline pH 7.4 + 0.05% Tween 20). The plate was blocked with 5% skim milk at 37 °C for 2 h. The polyclonal antibodies of TcdA and TcdB (chicken IgY; 1:1000 dilution; List Biological laboratories) were used for first hybridization; then, goat anti-chicken IgY secondary antibody, (HRP-conjugated; 1:5000 dilution; Invitrogen) was used. The TMB chromogenic reagent kit (Sangon, China) was used to determine the absorbance at 450 nm. Pure toxin A and toxin B (List Biological laboratories) were used as positive controls, and the standard curves were obtained using 20, 2, 0.2, 0.02, and 0.002 ng/ml of TcdA and TcdB [19 (link)].