Horseradish peroxidase hrp conjugated goat anti rabbit igg h l

For the focus-forming assay (FFA), all SARS-CoV-2 live virus infection experiments were performed in a biosafety level 3 (BSL3) laboratory. Vero E6 cells were seeded onto 96-well plates overnight and grown into confluent monolayers. Fifty microliters of 10-fold-diluted SARS-CoV-2 stock or supernatant of lung homogenate was added into a 96-well plate and adsorbed at 37°C for 1 h with agitation every 10 min. Then, the virus or supernatant of lung homogenate was removed and covered with 100 μL minimum essential medium (MEM) containing 1.2% carboxymethylcellulose (CMC). Twenty-four hours postinfection, the overlay was discarded and the cell monolayer was fixed with 4% paraformaldehyde solution for 2 h at room temperature. After being permeabilized with 0.2% Triton X-100 for 20 min at room temperature, the plates were sequentially stained with cross-reactive rabbit anti-SARS-CoV-N IgG (Sino Biological, Inc.) as the primary antibody and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch) as the secondary antibody at 37°C for 1 h. The reactions were developed with KPL TrueBlue peroxidase substrates. The numbers of SARS-CoV-2 foci were calculated using CTL ImmunoSpot S6 Ultra reader (Cellular Technology, Ltd.), and titers of the virus were expressed as focus-forming units (FFU) per milliliter.

For the focus-forming assay (FFA), all SARS-CoV-2 live virus infection experiments were performed in a biosafety level 3 (BSL3) laboratory. Vero E6 cells were seeded onto 96-well plates overnight and grown into confluent monolayers. Fifty microliters of 10-fold-diluted SARS-CoV-2 stock or supernatant of lung homogenate was added into a 96-well plate and adsorbed at 37°C for 1 h with agitation every 10 min. Then, the virus or supernatant of lung homogenate was removed and covered with 100 μL minimum essential medium (MEM) containing 1.2% carboxymethylcellulose (CMC). Twenty-four hours postinfection, the overlay was discarded and the cell monolayer was fixed with 4% paraformaldehyde solution for 2 h at room temperature. After being permeabilized with 0.2% Triton X-100 for 20 min at room temperature, the plates were sequentially stained with cross-reactive rabbit anti-SARS-CoV-N IgG (Sino Biological, Inc.) as the primary antibody and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch) as the secondary antibody at 37°C for 1 h. The reactions were developed with KPL TrueBlue peroxidase substrates. The numbers of SARS-CoV-2 foci were calculated using CTL ImmunoSpot S6 Ultra reader (Cellular Technology, Ltd.), and titers of the virus were expressed as focus-forming units (FFU) per milliliter.