Fig. 6

Neutralizing activity and epitope mapping of the identified neutralizers in an IgG format. (a) Neutralizing activity assay. Vero cells were co-cultured with the indicated concentrations of the purified IgGs of Clone#3 and #7 in the presence of 10 pg/mL TcdB for 24 h. E3 was included as a positive control. The percentage of rounded cells was calculated under a phase contrast microscope. (b) Epitope mapping. Dot blot was performed to identify the epitopes. 200 ng of each chimeric protein was loaded onto the nitrocellulose membrane (Bio-Rad, Cat. RAD-1,620,146). The membranes were then incubated with the purified IgG antibodies of Clone#3 and #7 at a final concentration of 1ug/mL, followed by secondary goat anti-human Fc-HRP (SouthernBiotech, Cat. 2048-05) (1:2000). The image was taken by G-Box Chemi system (Synoptics Ltd, Cambridge, UK)