SARS-CoV-2 NP and SP Antigen Production

The production and purification of SARS-CoV-2 NP and SP antigens were performed according to previously described protocols (14 (link), 24 (link), 25 (link)). The RBD of the SP was produced in Expi293F cells as previously described (24 (link), 25 (link)). Rabbit antisera against the RBD and NP were generated at BioGenes GmbH (Berlin, Germany) as follows: day 0 initial dose of 150 μg, day 7 booster of 75 μg, day 14 booster of 75 μg, day 28 booster of 150 μg, and day 42 final bleed. For affinity purification, the RBD and NP were coupled to CNBr-Sepharose 4B (Cytiva) according to the manufacturer’s protocol. The respective antisera were passed through coupled Sepharoses packed into Poly-Prep chromatography columns (Bio-Rad), washed with 20 column volumes of PBS, eluted (0.1 M glycine, 150 mM NaCl [pH 2.5]) with 2 M Tris (pH 9.0), concentrated using an Amicon Ultra 15-ml 100-kDa-NMWL (nominal molecular weight limit) centrifugal filter (Millipore/Merck), and dialyzed against PBS using Slide-A-Lyzer dialysis cassettes (Thermo Scientific).