SARS-CoV2 Human Neutralizing mAb Antibody

Microtiter wells were coated with 100 μL of ACE2-Fc (Cat# orb1945965) at 2 μg/mL in PBS at 4?C overnight. The wells were washed with PBS and blocked with 200 μL of 1% BSA/PBS. The antibody was titrated from 1 μg/mL in 1% BSA/PBS and mixed with equal volume of spike trimer Avi-tag of each variant. The blocker was discarded, and the wells incubated with 100 μL of the mixture of antibody and the spike trimer at 37?C for 1 hour. The wells were washed with PBS and the bound spike trimer was detected with 100 μL of High Sensitivity Streptavidin-HRP (Thermo Fisher 21130) (1:5, 000 in 1% BSA/PBS) at 37?C for 1 hour. The wells were washed with PBS and the wells were developed with 100 μL of MB/E Ultra Sensitive, Blue, Horseradish Peroxidase Substrate at 37?C for 15 min. The reaction was stopped with 100 μL of 0.6N H2SO4 and the signals were read at 450 nm using a plate reader. The experiments were performed in triplicates and the average was used to calculate % inhibition and IC50 (ng/mL) for each variant.