RBD-ACE2 competitive binding assay
RBD
VIRUS
The RBD neutralizing antibody against the different SARS-CoV-2 variants was evaluated using a commercial ELISA kit (Vazyme) by a surrogate virus neutralization test (sVNT). Sera are serially diluted by 3-fold from 1:5 to 1:1215 or 1:10935 with horseradish peroxidase-labeled recombinant RBD (HRP-RBD) protein and incubated at 37 °C for 30 minutes. HRP-RBD without the serum were added in duplicated in each plate as the negative control. Then 100 μl of dilution mixture was transferred to the corresponding wells of microplate that precoated hACE2 protein. After the incubation at 37 °C for 20 minutes, each well was washed with 350μl of wash buffer for 4 times. 100 μl of TMB substrate was added and incubated for 15 minutes at 37 °C and 100 μl of stop solution was used to stop the reaction, immediately followed by the plate reading at 450nm. The antibody titer was calculated as the reciprocal of the dilution for which the OD value was reduced by 50% of that of the negative control (IC