Pure toxin a and toxin b

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)].

TcdA and TcdB toxins of the infected tree shrew stools were detected by ELISA method. The fresh fecal samples were collected after C. difficile infection for one week of all animals. Feces were suspended to 0.5 g/ml concentration by using PBS buffer (pH 7.4). The fecal suspensions were coated in 96-well plate at 4 °C overnight, and then washed with PBST (PBS 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 (List Biological Laboratories, Chicken IgY, 1: 1000 dilution) were used for first hybridization; then Goat anti-Chicken IgY Secondary Antibody, HRP (Invitrogen, 1: 5000 dilution) was used. ELISA-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 determined at 20 ng/ml, 2 ng/ml, 0.2 ng/ml, 0.02 ng/ml and 0.002 ng/ml both for TcdA and TcdB.