FIG. 6.

Comparative antibody recognition of GNA lectin- or cellular CD81-bound E2. MAbs specific for the C-terminal epitope tag (11/4b), the HVR (6/16, 6/82a, and 7/59), and conformational epitopes (H53 and H60) are differentially shaded for ease of distinction. (A) Various concentrations of MAbs were evaluated for the ability to bind GNA lectin-captured soluble E2₆₆₁ antigen. Bound MAbs were visualized with antispecies IgG-HRP and the substrate tetramethylbenzidine. Results are shown as the mean optical density at 450nm (OD_450nm; based on three replicates, where the standard error in all cases was <5%) and represent a MAb concentration of 10 μg/ml. The relative affinities of the MAbs, i.e., the MAb concentrations (micrograms per milliliter) resulting in half-maximal binding, are shown. NS denotes that the MAb did not saturate the antigen. The MAbs are listed according to their epitope(s) within the E2 gp, where regions A to D are shown as depicted in Fig. 5. (B) A single concentration of each MAb (10 μg/ml) was tested for its ability to recognize E2₆₆₁ bound to the surface of RBL-CD81 cells. Bound MAb was visualized with antispecies IgG-PE and FACS analysis. Control MAbs include MAb 11/4b, specific for the C-terminal epitope tag known to be exposed on CD81-bound E2₆₆₁, and MAb V3, an irrelevant MAb which does not recognize E2₆₆₁ antigen. Results are expressed as the median FI. E2₆₆₁ and mock antigen failed to bind the parental RBL cells in the same experiment, giving a background median FI of 5.2. The data shown are representative of three experiments. (C) E2₆₆₁ antigen was incubated with each MAb (10 μg/ml), and the complexes were tested for the ability to bind RBL-CD81 cells. Results are expressed as the median FI of cell-bound MAb-E2 complexes. E2₆₆₁ and mock antigens failed to bind the parental RBL cells in the same experiment, giving a background median FI of 5.9. All MAbs were evaluated for their binding to RBL-CD81 cells, in the absence of E2 antigen, and gave values equivalent to background. The data shown are representative of three experiments.