FIG 6.

Epitope mapping: characterization of MAb CHL27. (A) SPR analysis of MAb binding to gH/gL. Procotol is as outlined in Fig. 5B. (B) Peptide ELISA using MAb CHL27. gH2 peptides were biotinylated at the N terminus, which facilitated binding to a 96-well streptavidin-coated plate. Each well was probed with CHL27 and visualized with goat anti-mouse IgG-horseradish peroxidase. (C) N-terminal amino acid sequence of gH2, with amino acid numbers shown below. The signal sequence (residues 1 to 18) and the CHL27 binding region (residues 37 to 47) are indicated with underlines. The sequence deleted in the gH2Δ48 construct is indicated with a bracket above the sequence. gH residues highlighted in green are important for CHL27 binding, as shown in (D). (D) Western blotting. Cells were transfected with the desired plasmids and cell lysates were separated by electrophoresis via SDS-PAGE. Lysates from cells transfected with empty plasmid (vector) and gL only served as negative controls. Antibodies used during Western blotting are shown to the right of each blot (anti-gH/gL PAb R176 and MAb CHL27).