FIG. 5.

Mapping of viral epitopes interacting with HS using an E2-heparin binding assay. (A) Inhibition of E2-heparin binding by monoclonal anti-E2 antibodies. E2 (1 μg/ml) was preincubated with the anti-E2 MAbs or IgG (50 μg/ml) for 1 h at 37°C and then added to ELISA plates coated with heparin (10 μg/ml). Heparin-bound E2 was detected using a polyclonal anti-E2 rabbit serum and colorimetric reaction as described in Materials and Methods. Results are shown as percent inhibition of E2-heparin interaction. Data are shown as mean percent inhibition of E2-heparin binding ± standard deviation (SD) obtained from three independent experiments. (B) Concentration-dependent inhibition of E2-heparin binding by anti-E2 antibodies. Recombinant E2 protein (1 μg/ml) was incubated with anti-E2 monoclonal antibody 2F10 (squares) or 49F3 (diamonds) or with control IgG (open triangles) at various concentrations for 1 h at 37°C. E2-antibody complexes were added to heparin immobilized on plates, and the E2-heparin interaction was analyzed as described above. The OD of the colorimetric reaction is proportional to heparin-bound E2. Results are presented as mean OD ± SD of a representative experiment performed in triplicate.