Horseradish peroxidase labeled goat anti mouse immunoglobulin

Baloxavir susceptibilities were determined by using a focus reduction assay as previously described [1 (link)] in humanized MDCK cells (i.e., hCK cells), which express high levels of α2,6-sialoglycans and very low levels of α2,3-sialoglycans [15 (link)]. hCK cells were kindly provided by Dr. Yoshihiro Kawaoka (University of Wisconsin–Madison). hCK cells in 96-well plates were infected with 1000 focus-forming units (FFU)/well of viruses. Virus adsorption was carried out for 1 h at 37 °C and then an equal volume of 1.2% Avicel RC-581 (DuPont Nutrition USA, Wilmington, DE, USA) in culture medium containing serial dilutions (0.025–2500 nM) of baloxavir was added to each well in triplicate. The cells were incubated for 24 h at 34 °C and then fixed with formalin. After the formalin was removed, the cells were immunostained with a mouse monoclonal antibody against influenza A virus nucleoprotein (Merck KGaA, Darmstadt, Germany), followed by a horseradish peroxidase-labeled goat anti-mouse immunoglobulin (SeraCare Life Sciences, Milford, MA, USA). The infected cells were stained with TrueBlue Substrate (SeraCare Life Sciences) and then washed with distilled water. After cell drying, the focus numbers were quantified by using an ImmunoSpot S6 Analyzer, ImmunoCapture software, and BioSpot software (Cellular Technology, Cleveland, OH, USA). The results are expressed as IC₅₀ values.

The antiviral activity of baloxavir was also determined by using a focus reduction assay (Tilmanis et al., 2017 (link)) under Avicel overlays (Matrosovich et al., 2006 (link)). Confluent monolayers of MDCK cells in 96-well plates were inoculated with 100 μl of 1000 focus-forming unit (FFU)/well of viruses. Virus adsorption was carried out for 1 h at 37°C and then 100 μl of 1.2% Avicel RC-581 (FMC BioPolymer) in culture medium containing serial dilutions (0.025–2500 nM) of baloxavir acid was added to each well in triplicate. The cells were incubated for 24 h and then fixed with formalin. After the formalin was removed, the cells were immunostained with a mouse monoclonal antibody against influenza A or B virus nucleoprotein (Merck; MAB8251, MAB8661), followed by a horseradish peroxidase-labeled goat anti-mouse immunoglobulin (SeraCare) as previously described (Tilmanis et al., 2017 (link)). The infected cells were stained with TrueBlue Substrate (SeraCare) and then washed with distilled water. After drying, the number of FFU was quantified by using an ImmunoSpot S6 Analyzer, ImmunoCapture software, and BioSpot software (CTL) as previously described (Tilmanis et al., 2017 (link); van Baalen et al., 2017 (link)). The IC₅₀ values were calculated by using GraphPad Prism.

Neutralization activities of SARS-CoV-2 were determined by using an FRNT as previously described^{14 (link)}. Serial dilutions of plasma were mixed with 1,000 focus-forming units of virus/well and incubated for 1 h at 37 °C. The antibody-virus mixture was inoculated on VeroE6/TMPRSS2 cells in 96-well plates in duplicate and incubated for 1 h at 37 °C. An equal volume of 1.2% Avicel RC-581 (DuPont Nutrition USA) in culture medium was added to each well. The cells were incubated for 24 h at 37 °C and then fixed with formalin. After the formalin was removed, the cells were immunostained with a mouse monoclonal antibody against SARS-CoV-1/2 nucleoprotein [clone 1C7C7 (Sigma-Aldrich)], followed by a horseradish peroxidase-labeled goat anti-mouse immunoglobulin (SeraCare Life Sciences). The infected cells were stained with TrueBlue Substrate (SeraCare Life Sciences) and then washed with distilled water. After cell drying, the focus numbers were quantified by using an ImmunoSpot S6 Analyzer, ImmunoCapture software, and BioSpot software (Cellular Technology). The results are expressed as the 50% focus reduction neutralization titer (FRNT₅₀). The FRNT₅₀ values were calculated by using GraphPad Prism (GraphPad Software).