Horseradish peroxidase conjugated goat anti rabbit igg secondary antibody

Total protein extracts of 1.5 x 10⁷ trophozoites from non-transfected, empty vector or FLAG-tagged-gFlHb transfected cultures were suspended in 500 μL of PBS, 1.5% Triton X-100, and were lysed by sonication. Protein concentration was determined using a Pierce BCA Protein Assay Kit (Thermo Scientific) and the absorbance was measured at 562 nm in a microplate reader (BioRad). Then, 50 μg of protein samples were separated by SDS-PAGE in 10% polyacrylamide gels under denaturing conditions. Western blots were performed by electrophoretic transfer of SDS-PAGE-separated proteins onto nitrocellulose membranes for 1.5 h at 300 mA in the cold. Membranes were blocked for 1 h at room temperature with a solution 5% nonfat dried milk and 0.1% Tween 20 in TBS. The membranes were then incubated overnight at 4°C with mouse monoclonal antibody α-FLAG M2 (Sigma-Aldrich Co., St. Louis, MO, USA) at a 1:100 dilution and anti-actin at 1:1000 dilution, prepared in blocking solution. The membranes were then washed three times with 0.1% Tween 20 in TBS and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Promega) diluted 1:30000 in blocking solution, for 1 h at 37°C and constant shaking. Chemiluminescence detection was performed with an Amersham Western Blotting ECL detection kit (GE Healthcare) according to manufacturer´s instructions.

The titre of infectious virus in the culture medium was determined by the focus-forming unit (FFU) assay. Vero E6 cells were seeded in a 96-well plate. The infectious cell culture medium was used to infect the cells and the plates were incubated at 37 °C. After 8 h, cells were washed with PBS, fixed with 4% paraformaldehyde and permeabilized with methanol. SARS-CoV-2 foci were developed using anti-SARS-CoV-2 N antibody, followed by addition of horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody and TMB substrate (Promega). The plates were incubated in dark condition at room temperature for 30 min, washed and dried for observation under a light microscope. The number of SARS-CoV-2 positive foci was scanned and counted in each well using an ImmunoSpot reader (CTL, Shaker Height, OH).