Sars cov 2 s1 s2 ecd 40589 v08b1

IgG was purified from sera taken from mice immunized with SARS-CoV-2 spike protein (INO-4800) (24 (link)) using the Nab protein G spin kit (89949; ThermoFisher). Ninety-six-well half-area plates (Corning) were coated at room temperature for 8 hours with 1 μg/ml PolyRab anti-His antibody (PA1-983B; ThermoFisher), followed by overnight blocking with blocking buffer containing 1× PBS, 5% skim milk, 1% FBS, and 0.2% Tween 20. The plates were then incubated with 10 μg/ml of SARS-CoV-2 S1+S2 ECD (40589-V08B1; Sino Biological) at room temperature for 1– to 2 hours. Purified IgG was serially diluted 3-fold (starting concentration, 100 μg/ml) with PBS with 1% FBS and 0.2% Tween and premixed with ACE2-IgHu at a constant concentration of 0.1 μg/ml. The premixture was then added to the plate and incubated at RT for 1 hour. The plates were further incubated at room temperature for 1 hour with goat anti-human IgG-Fc fragment cross-adsorbed Ab (A80-340P; Bethyl Laboratories) at a 1:10,000 dilution, followed by ant addition of the TMB substrate (ThermoFisher), and then quenched with 1 M H₂SO₄. Absorbances 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.

Ninety-six-well half -area plates (Corning) were coated at room temperature for 8 hours with the 1 μg/ml PolyRab anti-His antibody (Ab) (catalog number PA1-983B; ThermoFisher), followed by overnight blocking with blocking buffer containing 1× PBS, 5% skim milk, 1% fetal bovine serum (FBS), and 0.2% Tween 20. The plates were then incubated with 10 μg/ml of SARS-CoV-2 S1+S2 ECD (40589-V08B1; Sino Biological) at room temperature (RT) for 1–2 h, followed by an addition of either ACE2-IgHu serially diluted 5-fold (starting concentration, 2 μg/ml) or ACE2-IgMu (10108-H05H; Sino Biological) serially diluted 5-fold (starting concentration, 50 μg/ml). Serial dilutions were performed using PBS with 1% FBS and 0.2% Tween and incubated at RT for 1 h. The plates with ACE2-IgHu were then incubated at RT for 1 h with goat anti-human IgG-Fc fragment cross-adsorbed Ab (A80-340P; Bethyl Laboratories) at a 1:10,000 dilution. Similarly, the plates with ACE2-IgMu were incubated with goat anti-mouse IgG H+L horseradish peroxidase (HRP) (A90-116P; Bethyl Laboratories) at 1:20,000 dilution. Next, 3,3',5,5'-tetramethylbenzidine (TMB) substrate (ThermoFisher) was added to both plates and then quenched with 1 M H₂SO₄. Absorbances 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.