Sars cov 2 rbd

All recombinant proteins used in this study were His-tagged for normalization. SARS-CoV-2 and SARS-CoV N proteins were purified from pcDNA3.1-SARS-CoV-2 N or pDual-GC-SARS-CoV N transfected HEK293 T cells using Ni Sepharose affinity chromatography (GE Healthcare). SARS-CoV-2 S1 and SARS-CoV S1 were obtained from Sino Biological. SARS-CoV-2 RBD and SARS-CoV RBD were custom produced by GenScript. The amino acid sequence of these recombinant proteins are given in Supplementary Table 2. 25 μg of each antigen was coupled onto MagPlex-c microsphere (Luminex) using xMAP antibody coupling kit (Luminex) according to the manufacturer’s instructions. To assess the antibody reactivity, 1250 beads/antigen were incubated with various serum samples at 1:100 dilution in PBS containing 1% BSA for an hour at 37°C with agitation. The bound antibodies were detected with goat anti-human IgG, PE (eBioscience) at 1:1000 dilution. The level of antibody binding was determined using the Luminex MAGPIX system. The readings were normalised by dividing with the readings obtained from the anti-His tag antibody (Thermo Scientific) and presented as net mean fluorescence intensity (MFI).

SARS-CoV-2 RBD specific total antibody responses were determined using Sf9 insect cells SARS-CoV-2 spike protein RBD (GenScript, NJ, USA) as coating antigen and goat anti-mouse IgG HRP conjugate antibody (Jackson ImmunoResearch, Westgrove, PA, USA) as the detection antibody. The endpoint titers were determined by following the method, as described previously [16 (link)]. The cells secreting mouse IFN-γ by SARS-CoV-2-specific cells were evaluated using an ELISpot test for mouse IFN-γ (Mabtech, Stockholm, Sweden) by following the protocol as described previously [16 (link)], and the results are given in terms of spot-forming cells (SFCs)/10⁶ splenocytes.