GM1-ELISA

The ability of the CTB component in CWAE to bind to its cellular receptor was assessed by GM1-ELISA as previously described (Areas et al., 2004). Briefly, ELISA plates were coated with 1 μg/well GM1 ganglioside or BSA for 24 h. After washing, ELISA plates were blocked by incubating with 5% (m/V) skim milk for 2 h. The CWAE, CTB-UE, CTB or UreB proteins (100 μg/ml) were then added to ELISA plates and incubated for 2 h. After that, a proper dilution of anti-CTB polyclonal antibody (Biomade Technology) was added to the plates and incubated for 1 h. After washing, HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, USA) was added to the plate and incubated for 1 h. Substrate tetramethylbenzidine (TMB, Tiangen Biotech) was then added and incubated for 10 min. The absorbance was measured at 450 nm.