Ebov gpδtm

This assay utilizes THP-1 cells, human-derived monocytes, that reproduce phagocytic activity of primary monocytes but have divergent cytokine profiles on stimulation. Recombinant EBOV GPΔTM (Mayinga strain; IBT Bioservices) was biotinylated using LC-LC-Sulfo-NHS Biotin (ThermoScientific). Excess biotin was removed using a Zeba desalting column (ThermoScientific). Biotinylated GP antigen was then coupled to 1μm yellow-green Neutravidin beads (ThermoScientific) by incubating beads and antigen overnight at 4°C. Beads were washed twice with PBS containing 0.1% bovine serum albumin (BSA). Samples were diluted 1:100 in culture medium and incubated with GP-coated beads for 2h at 37°C. Unbound antibodies were removed by centrifugation prior to the addition of THP-1 cells at 2.5×10⁴cells/well. Cells were fixed with 4% paraformaldehyde and analyzed by flow cytometry. A phagocytic score was determined using the following formula: (percentage of FITC+ cells)*(geometric mean fluorescent intensity (gMFI) of the FITC+ cells)/10,000.

For the affinity ELISA, Costar half-area 96-well plates were coated with 30 μl of 1.25 μg/ml of recombinant EBOV GPΔTM (IBT Bioservices) overnight at 4°C. The plates were then blocked with 100 μl of PBS 5% skim milk (BD) for 1 hour at 37°C. Two-fold serial dilutions of the test antibodies (30 μl) in PBS 2% skim milk was added and incubated at 37°C for 2 hours. The plates were washed with 4 x 150 μl of PBS 0.1% Tween 20 with a plate washer (BioTek). The secondary antibody, goat anti-human IgG (H + L) -HRP (KPL), was added (30 μl; in PBS 2% skim milk) at a concentration of 0.5 μg/ml for 1 hour at 37°C. The plates were washed again and 50 μl of TMB Single Solution (ThermoFisher) was added. The plates were incubated in the dark at room temperature for 30 minutes and the absorbance was read at 650 nm on a BioTek Synergy HT plate reader (BioTek). Wells where the absorbance was reported as “OVRFLW” were discarded from the analysis. At every incubation step, the plates were sealed with a new plate sealing film (Excel Scientific). The binding affinity results are present in the supplementary Figure S1.