2.4 Enzyme-linked Immunosorbent Assay (ELISA)
ELISA was used to detect HA or SARS-CoV-2 Omicron BA.5 S-RBD-specific IgG, IgG1 and IgG2a antibodies in mouse sera. ELISA plates were pre-coated with 2 µg/mL HA or SARS-CoV-2 Omicron BA.5 S-RBD protein overnight at 4°C. The plates were washed with PBS containing 0.05% Tween-20 (PBST) and then blocked with PBS containing 2% skimmed milk powder at 37°C for 1 h. After washing the plates with PBST, serially diluted mouse serum was added into the plates, followed by incubation at 37°C for 1 h. After again washing with PBST, HRP-conjugated goat anti-mouse IgG, IgG1 or IgG2a was added to the plates, followed again by incubation at 37°C for 1h. After washing, TMB was added to the plates. Finally, 1N H2SO4 was added to the plates to terminate the reaction, and the absorbance value was measured at 450nm by microplate reader. Endpoint titers were expressed as the highest reciprocal serum dilution exhibiting an absorbance of 450 nm > 2.1-fold over the background values.
2.5 Hemagglutination Inhibition (HAI) Assay
HAI of immunized mouse sera was performed as previously described.14 First, serum samples were treated with receptor-destroying enzymes (Denka Seiken) overnight at 37°C and heat-inactivated at 56°C for 30 min. Second, 25 µL of 8 HAU of A/California/07/2009 (H1N1) virus were added to an equal volume of two-fold serially diluted mouse sera in wells of a U-bottom 96-well microtiter plate and incubated for 45 min to 1 h. Finally, 0.55% turkey erythrocytes were added into the wells, followed by further incubation at room temperature for 30 min. HAI titers were defined as the reciprocal of the highest serum dilution required for complete hemagglutination inhibition.