SARS-CoV-2 Antibody Binding Assay

Measurements of binding antibody levels to various SARS-CoV-2 strains were performed on sera from immunized mice, as previously described [22 (link)]. Briefly, ELISA plates were coated with 0.5 µg/ml of S protein purchased from ACRO Biosystems (Alpha strain, SPN-C52H6; Delta strain, SPN-C52He; Omicron BA.5, SPN-C522e) and incubated at 4 °C overnight, followed by blocking with PBS containing 1% bovine serum albumin (BSA) at RT for 2 h. After blocking, the wells were washed twice with PBS. Then, sera that were serially diluted with 1% BSA in PBS were added into each well in triplicate, and the plate was incubated at room temperature for 1 h. After an incubation period, the plates were washed three times with PBS containing 0.1% Tween-20 (PBST0.1) and then incubated for 1 h with peroxidase-affinipure goat anti-mouse IgG (H + L) (Jackson ImmunoResearch) (1:5000 dilution). After three washes with PBST0.1, signal was produced using 3,3’5,5’-Tetramethylbenzidine (TMB) substrate (TMBW-1000-01, SURMODICS). Finally, the reaction was stopped with 3 N HCl, and absorbance was measured at 450 nm by an ELISA reader (Versa Max Tunable Microplate Reader; Molecular Devices).