2.6. GP-NPC1 binding assay

Vero E6/NPC1-KO cells and Vero E6/NPC1-KO cells expressing HA-tagged NPC1 (Kondoh et al., 2018) were seeded in T-75 flasks. After harvesting with trypsin, the cells were sedimented at 1000 rpm for 5 min. CHAPS-NTE buffer (0.5% wt/vol CHAPS, 140 mM NaC1, 10 mM Tris-HC1, 1 mM EDTA; pH 7.5) (Miller et al., 2012) was added to cells to a final concentration of 10⁷ cells/ml. Then, EDTA-free Complete Protease Inhibitor Cocktail (Roche) was added. The cells were sedimented at 10,000×g for 10 min at 4 °C and the supernatant was harvested. VLPs (1 mg/ml in PBS) were treated with thermolysin (Sigma) at 37 °C for 30 min. The VLP solution was diluted at 1:10 with 0.05 M carbonate buffer (pH 9.6). ELISA plates (Nunc, Maxisorp) were coated with the diluted VLPs, and incubated at 4 °C overnight. The VLPs were removed and the plates were blocked with BSA (10 mg/ml in PBS) and incubated at room temperature for 2 h. Recombinant monoclonal antibody mAb114, which blocks binding of NPC1 to GP via interaction with the glycan cap and the inner chalice of GP, was generated as a positive control based on the sequence described previously using γ1HC, and κLC vectors (Cagigi et al., 2018; Saito et al., 2019; Tiller et al., 2008). Serially diluted HUP2976 and mAb114 (in PBS) were mixed with cell lysates (diluted at 1:20 with CHAPS-NTE buffer) and incubated at room temperature for 10 min. After washing the plates with 0.05% Tween 20 in PBS (PBST), the mixture was added to each well and incubated at 4 °C overnight. After removal of the mixture, the plates were washed with PBST 3 times, and rat anti-HA antibody 3F10 (Sigma) diluted with PBST containing BSA (5 mg/ml) was added, followed by incubation at room temperature for 1 h. After washing 3 times with PBST, horseradish peroxidase (HRP)-conjugated anti-rat IgG (H + L) (Jackson ImmunoResearch) was added to each well. After incubation at room temperature for 1 h, the plates were extensively washed and a 3,3’,5,5’-tetramethyl-benzidine (TMB) substrate (Sigma) was added, followed by incubation in the dark at room temperature for 30–60 min. The optical density (OD) value at 450 nm was measured after stopping the reaction with 1M phosphoric acid.