Polyrab anti his antibody

96-well half area plates (Corning) were coated at room temperature for 3 h with 1 μg/mL PolyRab anti-His antibody (ThermoFisher, PA1-983B), followed by overnight blocking with blocking buffer containing 1x PBS, 5% SM, 1% FBS, and 0.2% Tween-20. The plates were then incubated with 10 μg/mL of His6x-tagged SARS-CoV-2, S1+S2 ECD (Sino Biological, 40589-V08B1) at room temperature for 1-2 h. NHP sera (Day 0 or Week 6) was serially diluted 3-fold with 1XPBS containing 1% FBS and 0.2% Tween and pre-mixed with huACE2-IgMu at constant concentration of 0.4ug/ml. The pre-mixture was then added to the plate and incubated at room temperature for 1-2 h. The plates were further incubated at room temperature for 1 h with goat anti-mouse IgG H⁺L HRP (A90-116P, Bethyl Laboratories) at 1:20,000 dilution followed by addition of one-step TMB ultra substrate (ThermoFisher) and then quenched with 1M H₂SO₄. Absorbance at 450nm and 570nm were recorded with BioTEK plate reader.

96-well half area plates (Corning) were coated at room temperature for 8 h with 1ug/mL PolyRab anti-His antibody (ThermoFisher, PA1-983B), followed by overnight blocking with blocking buffer containing 1x PBS, 5% skim milk, and 0.1% Tween-20. The plates were then incubated with 1ug/mL of his-tagged BG505.MD39 at room temperature for 1 h. HIV antibodies were serially diluted 3-fold (starting concentration,100 ug/mL) with blocking buffer and incubated on the plates for 2 h. A no competitor control used dilution buffer at this step. C24 and C33 antibodies were biotinylated using Novus Biologicals Lightning-Link rapid type A Biotin antibody labeling kit (NovusBio, 370-0010) according to protocol. The biotinylated antibodies were added to wells at a concentration of 1 ug/ml (for C24) or 10 ug/mL (for C33) and incubated at RT for 1 h. The plates were further incubated at room temperature for 1 h with native streptavidin-HRP (Abcam, ab7403) at 1:15,000 dilution, followed by the addition of TMB substrate (ThermoFisher) and then quenched with 1 M H2SO4. Absorbance at 450 nm and 570 nm were recorded with a BioTek plate reader. Four washes were performed between every incubation using PBS with 0.05% Tween.