Hrp conjugated goat antimouse or goat antimonkey secondary antibody

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.

The six recombinant Gv_RBD proteins were diluted to 5 µg mL⁻¹ with coating buffer and coated on high‐binding 96‐well plates (Corning) overnight at 4 °C, respectively. After washing with PBS three times, the plates were blocked with 5% non‐fat milk/PBS for 1 h at room temperature. The plates were washed three times with PBS/T (containing 1% Tween‐20) again, and the animal serum or BALF was diluted serially in PBS, followed by incubating the plates for 1 h at 37 °C. After washing with PBS/T, HRP‐conjugated goat anti‐mouse or goat anti‐monkey secondary antibody (Invitrogen) was added at a dilution of 1:4000 to detect antigen‐specific lgG antibody in serum of BALB/c, K18 mice, or rhesus macaques. Correspondingly, the antigen‐specific IgA antibody in BALF was detected by adding HRP‐conjugated goat anti‐mouse secondary antibody with a dilution of 1:1000. After incubating for another 1 h, the plates were washed with PBS/T. Subsequently, 50 µL HRP substrate TMB solution (eBioscience) per well was added under dark, and the reaction was terminated with stop solution (Solarbio) after sufficient development. The absorption was measured at 450 nm. GraphPad Prism 8.0 software was used to perform non‐linear regression analysis on the data to calculate the endpoint titer.