Fig. 1

Results of in vitro characterizations of MAbs. (A) ELISA of Mabs. Hybridoma cell culture supernatants containing MAbs were tested against partial capsid protein p239 (HEV-3) coating antigen for their presence of capsid targeting MAbs. Two in-house MAbs, 7C3 and 6A2, that are directed against the HEV capsid protein, were used as positive controls (data unpublished). (B) Western blot analysis for determining the specificity of MAb supernatants against p239 protein. To evaluate the specificity of multiple MAbs simultaneously, the membrane was split and processed separately for each Mab, then reassembled for imagine (C) Peptide ELISA of MAbs in cell culture supernatants. Overlapping peptides, covering the complete HEV p239-region, were used as coating antigens. Two in-house MAbs, 7C3 and 6A2, which are directed against the HEV capsid protein, as well as serum of a rabbit that was vaccinated with partial capsid protein p429 (Supplemental Data 1) with detection reagent, were used as positive controls. (D) Immunofluorescence assay of supernatants in non-transfected mock control (top) and pVax1-ub HEV-SMP transfected cells (bottom). Two in-house MAbs, 7C3 and 6A2, that are directed against the HEV capsid protein, were used as positive controls. (E) Immunofluorescence staining of cells transfected with Kernow C1 P6 and pUC83-2 in a concentration of 5 µg/ml. The analysis was performed using an ELISpot Reader (Immunospot, Shaker Hights, Cleveland, UA). A monoclonal anti-HCV antibody was used as a negative control. (F) Neutralization activity of monoclonal antibodies against non-enveloped HEV-3 strain Kernow C1 p6 in serial dilutions, starting at a concentration of 5 µg/ml. An anti-HCV antibody and phosphate buffered saline (PBS) were used as negative controls. Data were normalized to corresponding PBS control. Depicted are the means and standard error of measurements.