A. E5/G6 epitope analysis of TPMV N. Using a variety of synthesized 10-mer peptides, we confirmed E5/G6 reactivity against TPMV sequence. Further, we determined which amino acid changes in this region were essential for E5/G6 binding. The peptide changes at positions 178 (R→V), 182 (K→R), 183 (Q→K), and 184 (H→P) were sufficient for MAb E5/G6 binding. B. Insertion of several amino acid mutations changing E5/G6 binding. C. Confirmation of the antigenicitiy of each baculovirus-infected High Five cell antigens expressed by recombinant baculoviruses. The rN antigen having the original sequence (rN/wt) reacted with immune serum but not with MAb E5/G6. On the other hand, the rN with the E5/G6 epitope (rN/E5G6) reacted with immune serum, as well as MAb E5/G6. D. Western blotting analysis using sera from rabbits immunized with E. coli-expressed rN antigens. Both rN antigens (rN/wt and rN/E5G6) were detected by a band of about 50 kDa, which corresponded to authentic TPMV N. Lane 1, rN/wt; lane 2, rN/E5G6; lane 3, TPMV-infected Vero E6 cells.