Article Figures & Data
Figures
- Fig. 1.
Indirect immunofluorescence staining of full-length PORF2 protein with MAbs against PORF2. HepG2 cells were transfected with pCI-ORF2 for 40 h, fixed, and stained with MAbs followed by fluorescein-conjugated anti-mouse IgG (green) together with propidium iodide to counterstain nuclei (red). Transfected cells were stained with HEV-specific MAb 1E6 (A), 4B2 (B), or 2E2 (C) or with irrelevant MAb 1H1 (D). Magnification, ×200.
- Fig. 2.
Endpoint titration of MAb reactivities against GST-ORF2.1 antigen (A) and VLP antigen (B). ELISA plates were incubated with the indicated concentrations of each MAb for 1 h, and bound antibody detected with HRPO-conjugated anti-mouse IgG. 1H1, irrelevant MAb.
- Fig. 3.
Mapping of MAb epitopes within PORF2. Equimolar amounts of fusion proteins (GST with the indicated fragments of PORF2) were separated on SDS-PAGE gels and transferred to nitrocellulose membranes, and each membrane was reacted with a single MAb. Immune complexes were detected by enhanced chemiluminescence. (A) MAb 4B5 and schematic of truncated PORF2 fragments in each lane. (B) MAb 2E2; (C) MAb 4B2; (D) MAb 1C7; (E) MAb 3B2; (F) MAb 1E6; (G) MAb 1E7. Note the exclusive reactivity of 2E2 and 4B2 with the ORF2.1 fragment. Lane MW, size markers.
- Fig. 4.
Competition matrix between HEV-specific MAbs tested on GST-ORF2.1 (A) and VLP (B) ELISAs. ELISA wells were reacted with saturating amounts of the competing MAb before addition of biotin-labeled MAbs, the residual binding of which was then detected with HRPO-conjugated streptavidin and quantitated by comparison with dilutions of each biotin-labeled MAb alone. Symbols: □, <30%;
, 30 to 70%;, 70 to 90%; ■, >90% inhibition of binding. (C and D) Two-dimensional surface-like maps of epitopes on GST-ORF2.1 (C) and VLPs (D). Overlap indicates more than 30% inhibition of ELISA reactivity between pairs of MAbs for each antigen. - Fig. 5.
Competition between HEV-specific MAbs and convalescent-phase patient sera for binding to VLPs. VLP ELISA wells were reacted with saturating amounts of competing MAb before addition of patient sera, the binding of which was then detected with HRPO-conjugated anti-human IgG. Residual human anti-HEV binding was quantitated by comparison with a standard curve and calculated as a percentage of binding in the presence of irrelevant MAb 1H1. The mean and error of two separate experiments are shown. Solid bars, patient G31 (Nepal); open bars, patient G37 (Nepal); hatched bars, patient NIH116 (Mexico).
Tables
- Table 1.
Summary of MAb characteristics
MAb Isotype Epitopea PORF2 IFb Blocking (%)c 4B5 IgG1 394–414, conformational ++++ 0–4 3B2 IgG1 414–434 ++++ 0 1C7 IgG1 414–434 ++++ NT 1E6 IgG2b 434–457 ++++ 28–38 1E7 IgG2b 434–457 ++++ NT 4B2 IgG1 ORF2.1, conformational + 52–59 2E2 IgG1 ORF2.1, conformational +/− 60–76 a Epitope range in aa. Data are from Fig.3.
b Indirect immunofluorescence reactivity against HepG2 cells expressing full-length PORF2. ++++, very strong reactivity; +, weak reactivity; +/−, very weak reactivity. Data are from Fig. 1 and results not shown.
c Blocking of convalescent-phase patient IgG binding to VLPs: range between respective means for three patient sera. Data are from Fig. 5. NT, not tested.
