Goat anti human igg fc secondary antibody with hrp

First, 96-well Maxisorp plates (Thermo Fisher) were coated overnight at 4 °C with 2 µg/mL of NiV G or LayV G_SM in 50 mM Tris and 150 mM NaCl at pH 8. Plates were slapped dry, washed 3× in TBST, and blocked with Blocker Casein (ThermoFisher) for 1 h at 37 °C. Then, plates were slapped dry and washed 4× in TBST, and 1:4 serial dilutions of NHP sera were made in 50 μL TBST and incubated at 37 °C for 1 h. Plates were slapped dry and washed 4× in TBST followed by addition of 50 μL 1:5,000 Goat anti-Human IgG Fc Secondary Antibody with HRP (Invitrogen) for 1 h at 37 °C. Plates were slapped dry and washed 4× in TBST followed by addition of 50 μL TMB Microwell Peroxidase (Seracare). The reaction was quenched after 4 min with 1 N HCl and the A450 of each well was read using a BioTek Synergy Neo2 plate reader. Data were plotted and fit in Prism (GraphPad) using nonlinear regression sigmoidal, 4PL, X is log(concentration).

96-well Maxisorp plates (Thermo Fisher) were coated overnight at 4°C with 2 μg/mL of NiV G or LayV G central stalk stabilized ectodomain in 50mM Tris and 150mM NaCl at pH 8. Plates were slapped dry, washed 3X in Tris Buffered Saline Tween (TBST) and blocked with Blocker Casein (ThermoFisher) for 1 h at 37°C. Plates were slapped dry and washed 4X in TBST. 1:4 serial dilutions of NHP sera were made in 50 μL TBST and incubated at 37°C for 1 h. Plates were slapped dry and washed 4X in TBST followed by addition of 50 μL 1:5000 Goat anti-Human IgG Fc Secondary Antibody with HRP (Invitrogen) for one hour at 37°C. Plates were slapped dry and washed 4X in TBST followed by addition of 50 μL TMB Microwell Peroxidase (Seracare). The reaction was quenched after 4 minutes with 1 N HCl and the A450 of each well was read using a BioTek Synergy Neo2 plate reader. Data was plotted and fit in Prism (GraphPad) using nonlinear regression sigmoidal, 4PL, X is log(concentration).