Fig 1: The activity of the selected nanobody clones evaluated by indirect ELISA (A) and the virus-neutralizing activity assessed using rVSV-GP (B). (A) – High-binding Polystyrene Microtiter plates were coated with 100 µl (1 µg/ml) of EBOV GP (H. sapiens-wt/GIN/2014/Kissidougou-C15). On the next day, the wells were washed with 0.1% PBST five times and blocked with 5% non-fat skim milk in PBST. Different dilutions of the nanobodies in blocking buffer were added to the plates and incubated at 37°C for 1 h. The wells were washed five times, and HRP-conjugated Anti-Myc Tag antibodies (Abcam, UK, ab1326) in blocking buffer (1 : 5,000) were added for incubation at 37°C for 1 h. The wells were washed five times, TMB was added, and the results were evaluated. (B) – Dilutions of rVSV-GP (H. sapiens-wt/SLE/2014/Makona-G3735.1) in buffer (10 mM Tris-HCl, pH 7.5; 1mM EDTA, 10% sucrose) were prepared. A mixture of equal volumes of the nanobodies and virus stocks was incubated at 37°C for 1 h and then transferred to Vero E6 cell monolayers. After cell incubation with the nanobody + virus complex at 37°C for 2 h, the cells were coated with agar. The plates were incubated in 5% CO2 atmosphere at 37°C for 48 h. The results were evaluated by counting the number of plaques under the microscope. The assay was performed in triplicate. The following formula was used to determine the plaque-forming units (PFU) per milliliter: PFU/ml = (mean PFU count/0.2 ml) × dilution factor