Goat anti human igg fc hrp

The affinity of S-RBD or S1 for hACE2-Fc WT or variants was measured using a standard ELISA assay. Briefly, purified CoV2-S-RBD (2 μg/mL) or S1 (2 μg/mL) or the prefusion S-protein trimer (2 μg/mL) was coated onto 96-well ELISA plates (Fisher Scientific, #07-000-102) and incubated at 4 °C for 18 h. The coated plates were washed three times with 200 μL of PBST and then blocked with 200 μL of 3% BSA (Sigma-Aldrich # A8327) in PBST (Sigma-Millipore # 524653) and incubated for 1 h at room temperature. After washing the plates three times with 200 μL of PBST an increasing concentration of hACE2-Fc proteins were added and incubated for 1 h at room temperature. The unbound hACE2-Fc was removed by washing the plate three times with 200 μL of PBST. The bound hACE2 was detected using Goat-anti-human-IgG-Fc-HRP (Jackson Immuno Research # 109-035-008; 1:5000 dilution) using 50 μL TMB substrate (Pierce/ThermoFisher Scientific # 34028). After 3 min, the reaction was stopped using 50 µL of 2 N H₂SO₄. The optical density of the reaction was measured at 450 nm using a plate reader (Molecular Devices Gemini XPS). The data was analyzed and EC50 was calculate using Prism (GraphPad).

Purified inactivated CORAVAX or control vaccine were coated in 3-fold decreasing concentrations starting at 25 μg/mL. Control antigen RBD-His was coated in 3-fold decreasing concentrations, starting at 300 ng/well A constant amount of SARS COV/2 Anti-receptor binding domain (RBD) mouse IgG2a antibody (InvivoGen, Cat # srbd-mab10, 1 μg/mL) or the recombinant human ACE-2 Fc chimera (human IgG, Biolegend, Cat # 793208, 1 μg/mL) were added to the wells in 1% Milk in 1× PBST (0.05%Tween-20). Secondary antibodies, goat anti-mouse IgG-Fc HRP (Southern Biotech, Cat# 1033-05, 1:2000 in PBST) or goat Anti human IgG-Fc HRP (Jackson ImmunoResearch, Cat# 109-035-098, 1:2000 in PBST) were used to detect the bound RBD mouse IgG2a antibody and human ACE-2 Fc chimera (human IgG) protein respectively.
Optical density was measured at 490 nm and 630 nm using an ELX800 plate reader (Biotek Instruments, Inc., Winooski, VT). Background subtracted data (OD430 - 630 nm) were analyzed with GraphPad Prism (Version 6.0 g) using 4-parameter nonlinear regression.