Human monoclonal anti sars cov s cr3022

ELISA plates, Nunc MaxiSorp (Invitrogen), were coated with purified recombinant SARS-COV-2 RBD domain (BIR resources, NR-52306) at 2 μg/ul in PBS. Coating was carried out overnight at 4°C. Protein was blocked in 2% BSA, 1% tween-20 in PBS for 30 minutes at RT. The following anti SARS-CoV-2 S monoclonal and polyclonal antibodies were serially diluted by 2-fold dilutions in blocking buffer: mouse anti-SARS-CoV S monoclonal IgM 154C, mouse anti-SARS-CoV S monoclonal IgG2a 240C, mouse anti-SARS-CoV S monoclonal IgG2a 341C, mouse anti-SARS-CoV S monoclonal IgG2a 540C, human monoclonal anti-SARS-CoV-S CR3022 (BEI Resources). Human patient sera from a SARS-CoV-2 patient was used as a positive control. Dilutions ranged from1:10 to 1:10480, and were incubated for 1 hour at RT. Anti-mouse HRP, and anti-human-HRP secondary antibodies were used at 1:4000 concentration in blocking buffer, and were incubated 1 hour at RT. 50 μL of TMB HRP substrate (ThermoFisher Scientific) was added, and following incubation for 10 minutes at RT, 50 μL of 2N H2SO4 was added as a stopping solution. Plate absorbance at 405nm was measured using a CLARIOstar® Plus plate fluorimeter (BMG Labtech).