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.