Figure 2

Epitope Mapping of MAb 7C7. (A) Fragmentation of the VP2 protein into 7 continual overlapping segments. Western blotting of the fragments with anti-GST showed expression of all constructs, while blotting with 7C7 did not recognize fragments F and G, indicating that the epitope lies between amino acids 130 and 162 of VP2. (B) Fragmentation of amino acids 1-159 of VP2 into 6 continual overlapping segments. Again, blotting with anti-GST confirms protein expression, while blotting with 7C7 indicates that the epitope lies on fragment "d" between amino acids 145-159 of VP2. (C) Fragmentation of "d" into 6 continual overlapping peptides. Anti-GST blotting shows expression while the epitope of 7C7 is restricted to peptide "4" corresponding to amino acids EDSHPP. (D) Mutational analysis of the putative 7C7 epitope EDSHPP: Mutated epitopes were expressed as GST fusion proteins and expression was confirmed by Western blotting with anti-GST antibody. The minimal epitope EDSHP as well as the aspartic acid to asparagine substitution (ENSHPP) present in EV71 BrCr strain and CAV16 could be recognized by our MAb 7C7. However, the threonine to serine mutation (EDTHPP) of some C4 strains and the double mutation of human echoviruses (EDNAPP) could not be recognized.