SARS-CoV-2 Spike Protein Antibody Epitope Mapping

Antibody matched pair selection was performed using a set of HRP conjugated and unconjugated mAb in sandwich ELISA in the presence of recombinant (S) protein. The HRP-conjugation of one mAb (SpMA-01) was performed using EZ-link Plus activated peroxidase labeling kit (ThermoFisher Scientific, USA) and titrated in an indirect ELISA prior to use in epitope mapping (data not shown). Briefly, the 96-well microplate was coated with 50 µL of 2 µg/mL of unconjugated/capture antibodies (SpMA-02) in coating buffer (pH-9.6) and incubated overnight at 4°C. In the control wells, 50 µL purified normal mouse IgG (2 µg/mL) and 50 mM PBS were added. Next day the plate was washed with 50 mM PBS, and the non-specific sites were blocked with 1% milk protein in 50 mM PBS and incubated for 1hr at RT. After washing the plate, 50 µL of 1 µg/mL of (S) in 50 mM PBS was added to the wells and incubated for 1 hour at room temperature. The plate was washed with PBS-Tween-20, and the wells were incubated with 50 µL of optimum concentration of HRP-labeled detection antibody (SpMA-01) and incubated for 1 hour at room temperature. After washing the plates three times with PBS-T-20 (VWR, USA), 50 μL of TMB substrate was added to each well and incubated for 10 to 15 minutes. The plate was read at 450 nm in a Spectramax M-2 plate reader (Molecular Devices, USA) after adding 50 μL of stop solution (5 N sulfuric acid) to each well.