Fig. 6.

Fine mapping of the target epitope for E184L mAbs (2D2, 3H6, and 4C10) by sequential amino acid deletion from the N and C-terminus end of the peptide ¹⁴¹ILMVADNLYGEQDPTEFFSLIIEQTKTIKK¹⁷⁰. A) Analysis of the reactivity of the truncated peptides (R5 peptides) to 2D2, 3H6, and 4C10 mAbs by using Western blot. The MBP fused peptides were probed with each mAb separately. The three mAbs reacted to peptide R5–1 to R5–12 until the Aspartic acid at position 153 (D¹⁵³) was removed from the N terminus end, and to peptide R5–15 to R5–20 until the phenylalanine at position 158 (F¹⁵⁸) was removed from the C terminus end. These results indicate that the core amino acid for 2D2,3H6 and 4C10 mAbs lay between ¹⁵³DPTEFF¹⁵⁸. C) Dot blot assay to further confirm the linear core amino acid sequence of mAbs 2D2, 3H6 and 4C10. E184L-R5 peptides (R5–9 to R5–14 and R5–18 to R5–23) were expressed, spoted into nitrocellulose membrane and probed with 2D2,3H6 and 4C10 mAbs. The full length E184L and MBP pprotein were used as postive and negative control and are indicated as “PC” and “NC”, respectively. C) schematic design showing the sequential deletion of amino acid and its respective result of Western blot after staining with 2D2,3H6, and 4C10 mAbs. MBP antibody was used to test the successful expression of each peptide.