SARS2 Assay

Direct binding and sVNT assay.

For direct binding, hACE2 protein (GenScript) was coated at 100 ng/well in 100 mM carbonate-bicarbonate coating buffer (pH 9.6). HRP-conjugated SARS-CoV-2 N, S1, RBD or HRP-conjugated SARS-CoV-RBD (all purchased from GenScript) was added to the hACE2 coated plate at different concentration in OptEIA assay diluent (BD) for 1h at room temperature. Unbound HRP-conjugated antigens were removed by five phosphate buffered saline, 0.05% tween-20 (PBST) washes. Colorimetric signal was developed on the enzymatic reaction of HRP with chromogenic substrate, 3,3’,5,5’-tetramethylbenzidine (TMB) (Invitrogen). Equal volume of TMB stop solution (KPL) was added to stop the reaction, and the absorbance reading at 450 nm and 570 nm were acquired using Cytation 5 microplate reader (BioTek). For the surrogate neutralization test (sVNT), 6 ng of HRPRBD (from either virus) was pre-incubated with test serum at the final dilution of 1:20 for 1 h at 37°C, followed by hACE2 incubation for 1 h at room temperature. Inhibition (%) = (1 - Sample OD value/Negative Control OD value) x100.

Indirect ELISA.

SARS-CoV-2 and SARS-CoV N proteins were expressed from the pcDNA3.1 SARS-CoV- 2 N and pDualGC SARS-CoV N transfected HEK293T cells and purified using Ni Sepharose (GE healthcare). For indirect ELISA, 100 ng of each protein was coated onto MaxiSORP ELISA plate (Nunc) using 100 mM carbonate buffer and blocked with BD OptEIA (BD). COVID-19, SARS patient sera were tested at a dilution of 1:50 and detected by Goat-anti-human IgG-HRP (Santa Cruz) at 1:10,000 dilution. The chromogenic signal was developed using TMB substrate (Invitrogen) and the reaction was stop with TMB stop solution (KPL). Absorbance readings at 450 and 570 nm were obtained using Cytation 5 microplate reader (Bio-Tek).

Capture ELISA.

96-well Maxisorp plates (Nunc) were coated with 10 μg/ml of anti-human IgM (SeraCare) or anti-human IgG (Jackson labs) in bicarbonate buffer overnight at 4oC. Wells were blocked using BD OptEIA assay diluent (BD) for 1 h at 37oC and heat-inactivated sera diluted 1:50 were next added and incubated for 1 h at 37oC. Following extensive washing, SARS-CoV-2-HRP (GenScript) diluted 4 μg/ml was added and incubated for 30 min at 37oC. Chromogenic reaction was quantified following the addition of TMB substrate (Invitrogen) and stop solution (KPL SeraCare). The 11 absorbance of the samples was measured at 450 nm and the background at 570 nm. Negative controls consisted of 37 naïve human sera. Results are presented as fold-change over average reading of negative controls.