Sars cov 2 s1 s2 ecd

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 (Sino Biological, 40589-V08B1) at room temperature for 1 to 2 hours. ACE2-IgMu (10108-H05H; Sino Biological) 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 constant concentrations (ranging from 0.01 to 10 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 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.

Competitive inhibition of SARS-CoV-2
Spike binding to ACE2 receptor in the presence of SARS-CoV-2 Spike
mAb clones was evaluated by a competition ELISA as described.^{34 (link)} First, 96-well half-area plates (Corning, USA)
were coated at room temperature for 3 h with 1ug/mL SARS-CoV-2 S1+S2
ECD (Sino Biological, USA), followed by overnight blocking at 4 °C
with blocking buffer containing 1× PBS, 5% skim milk, and 0.1%
Tween-20. Plates were washed four times with wash buffer containing
1× PBS and 0.05% Tween-20. A huACE2-IgMu control (Sino Biological,
USA), PBS buffer control, or mouse hybridoma gradient purification
was serially diluted 3-fold with blocking buffer and incubated on
the plate for 1 h at room temperature (starting concentration 100
μg/mL for protein control and 1:100 dilution for mAb clones).
Plates were washed four times. Recombinant huACE2-IgHu was added at
a constant concentration of 0.1 μg/mL to each of the wells and
incubated for 1 h at room temperature. After washing four times, the
plates were further incubated at room temperature for 1 h with goat
anti-human IgG-Fc fragment cross-adsorbed antibody (Bethyl Laboratories,
USA) at 1:10 000 dilution. This was followed by four washes
and the addition of TMB substrate (ThermoFisher, USA). The plates
were then quenched with 1 M H₂SO₄. The absorbances
at 450 and 570 nm were recorded with a BioTek plate reader.