Quantifying Ebola Virus Attachment to NPC1

Plates were coated with GP-specific mAb KZ52 (44 (link)) diluted to 2 μg/ml in phosphate-buffered saline (PBS). VSV-GP mutants were normalized for GP content and then incubated at 37°C for 1 h in the presence of 250 μg/ml thermolysin (Sigma-Aldrich, St. Louis, MO), before the addition of 10 mM phosphoramidon to stop the reaction. ELISA plates were blocked using PBS supplemented with 3% bovine serum albumin (PBSA) (Thermo Fisher Scientific, Waltham, MA). THL-treated virus was added to blocked KZ52-coated plates and allowed to adsorb at 37°C for 1 h. Following washing with 3% PBSA, a dilution series of FLAG-tagged NPC1 domain C (45 (link)) was added and allowed to bind for 1 h at 37°C. Bound domain C was then detected using a horseradish peroxidase (HRP)-conjugated anti-FLAG antibody (Ab) (Sigma-Aldrich, St. Louis, MO) and the Ultra-TMB substrate (Thermo Fisher Scientific, Waltham, MA). Fifty percent effective concentration (EC₅₀) values were calculated using nonlinear regression of data from two independent experiments with three technical replicates each.