Antigen production for ELISA and ELISpot

For the production of HA-tagged EBOV-GP, sub-confluent T175 flasks of 293 T cells (human kidney cell line) were transfected with a eukaryotic expression vector (pDisplay) that expresses HA-tagged EBOV-GP and purified over a column. Fractions were collected and analyzed by Western Blot, and peak fractions were pooled and dialyzed against PBS in 10,000 molecular weight cutoff dialysis cassettes (Thermo Fisher Scientific) to remove excess HA peptide. After dialysis, the protein was quantitated by UV spectrophotometry and frozen in small aliquots at −80 °C. Stripped RABV Glycoprotein (G) antigen was produced by infecting BEAS-2B cells with rVSV-ΔG-RABV-G-GFP in OptiPRO SFM. Viral supernatants were concentrated and ultracentrifuged through a 20% sucrose cushion. Viral pellets were then resuspended in detergent-containing buffer and centrifuged to strip antigen from the virus. All antigens were further analyzed and characterized by SDS-PAGE gel and Western Blot.