P06dhurb

The trimeric spike protein of Wild, Delta, C.1.2, Omicron variants were coated on high binding 96 well plate (Biomat, MT01F4-HB8) at 0.1 μg per well using phosphate-buffered saline (PBS, pH-7.4) overnight at 4 °C. Blocked with 3% BSA in PBS-T for 2 h at room temperature and incubated with 100 μl of anti-RBD monoclonal antibodies (Invitrogen, P06DHuRb, twofold diluted in 1% BSA in DPBS-T from 2.5 to 0.02 µg/ml concentration) to spike variants coated wells for 1 h at 37 °C. All wells were washed and incubated 100 μl of goat anti-rabbit IgG-HRP antibody at dilution of 1 in 5000 in blocking buffer. The level of anti-RBD bound to each spike variants were estimated by 3, 3′, 5, 5′-Tetramethylbenzidine (TMB) substrate. The level of binding of IgGs in each sera sample to spike variants were quantified by measuring the optical density (OD) at 450 nm in i3x plate reader (Molecular Devices, San Jose, CA). OD of each sera sample for spike variants was subtracted to respective spike variants blank OD (without sera). The percentage of maximum anti-RBD antibody binding was calculated by the following formula: [OD of concentrations/OD of highest concentration of spike variants)] × 100%. The Half maximal effective concentration (EC₅₀) of each variant was calculated by agonist versus normalized response–variable slope model.