OC43 and NL63 RBD ELISA were carried out as previously described 80 (link),81 (link). Briefly, coating was performed by Streptavidin (Invitrogen) at 4 μg/mL in Tris-Buffered Saline (TBS) pH 7.4 for 1 h at 37°C followed by blocking with Non-Animal Protein-BLOCKER™ (GBiosciences). Then biotinylated spike RBD antigens for OC43 and NL63 were added at 1 μg/mL at 37°C for 1 h. All plasma samples were heat-inactivated before usage to minimize risk of residual virus in serum and then incubated at serial dilution followed by multiple washes and incubation with horseradish peroxidase-conjugated secondary Goat Anti-Human secondary IgG (Cat No: 109–035-008, Jackson ImmunoResearch) at 1:40,000 dilution in 3% milk at 37°C for 1 h. The resulting plate was washed and 3,3’,5,5’ -Tetramethylbenzidine (TMB) Liquid Substrate (Sigma-Aldrich) was added for optical density (OD) measurement at 405 nm after stopping the reaction with 50 μl of 1 N HCl.