Stop solution

The S1, S2, M, N and E proteins (same with the protein in Section 2.7) of the SARS-CoV-2 Wuhan-Hu-1 strain were utilized to coat 96-well ELISA plates (Corning, NY, USA) at a concentration of 0.1 μg per well and incubated overnight at 4 °C. Diluted serum and standard samples were added to 96-well plates precoated with specific antibody. After washing, horseradish peroxidase-conjugated antibody (Thermo Fisher, Waltham, MA, USA) and 3,3′,5,5′-tetramethylbenzidine reagent (Solarbio, Beijing, China) were added successively for signal development. The stop solution was then added (Solarbio, Beijing, China), and the absorbance at 450 nm was read with a microplate reader. The specific IgG titers were determined by end titration utilizing the reciprocal of the lowest serum dilution that produced an OD value 2.1-fold greater than that in the prebleed. In addition, commercialized anti-SARS-CoV-2 (Omicron B.1.1.529) antibody IgG titer serologic assay kit was used to test the S1-specific IgG titers (Acrobiosystems, Newark, DE, USA) according to the manufacturer’s protocol.

Recombinant proteins D614G_RBD, B.1.351_RBD, and B.1.617.2_RBD were diluted at one level at 5 µg mL⁻¹ concentration in coating buffer to be coated on 96 high‐bond well plates (Corning) overnight at 4 °C, respectively. After being washed three times with PBS, the plates were blocked with 5% nonfat milk/PBS for 1 h. After another series of washings, the immunized animal serum was serially diluted, added to each well in duplicate, and then incubated at room temperature for 1 hr. The detection of antigen‐specific IgG antibody in serum of BALB/c mice, hACE2 mice, or rhesus macaques was conducted through adding HRP‐conjugated goat antimouse or goat antimonkey secondary antibody (Invitrogen) respectively at dilution of 1:10 000 and incubating for another 1 h after washing with PBS/T (containing 1% Tween‐20) three times. The plates were washed four times before adding 100 µL of TMB (eBioscience) solution at each well. After 5 min of chromogenic progress at room temperature, a 100 µL stop solution (Solarbio) was added to quench the reaction followed by an absorption measure at 450 nm. The data were analyzed using GraphPad Prism 8.0 software for nonlinear regression to calculate endpoint titers.

To determine the RVFV Gn- and Gc-specific IgG, IgG1, or IgG2a titer in mice, RVFV Gn or Gc proteins were diluted in carbonate–bicarbonate buffer and coated in the 96-well high-binding microplates (Corning, NY, United States) with 0.2 μg/well at 4°C overnight. The plates were then blocked with PBS containing 2% BSA at 37°C for 1 h. After three rinses with PBST (PBS containing 0.2% Tween-20), serially diluted sera of mice in dilution buffer (PBST containing 0.2% BSA) were incubated with RVFV Gn or Gc proteins coated in the microplates at 37°C for 1 h. After that, the microplates were rinsed three times with PBST and incubated with the secondary antibodies at 37°C for 1 h, which target mouse IgG, IgG1, or IgG2a (Abcam, Cambridge, United Kingdom). After microplates rinsed three times, the TMB substrate solution (Solarbio, Beijing, China) was added in the microplates for 6 min at room temperature and the reaction was terminated by stop solution (Solarbio, Beijing, China). The optical density (OD) was measured at 450/630 nm (OD450/OD630; SPECTRA, Molecular Device, San Jose, CA, United States). We defined the endpoint titers as the reciprocal of the highest serum dilution, the OD value of which was 2.1-fold higher than that of the negative control.