Immulon 4 microtiter plates

These assays were done using methods similar to those described^{52 (link)} and Immulon 4 microtiter plates (VWR, Radnor, PA) coated with 100 ng per well of S1, S2, N, or RBD protein (SinoBiological #40592-VNAH). For IgA assays, a pooled rhesus serum collected day 14 after infection with SARS-CoV-2 was used as standard after depletion of IgG using GE Healthcare Protein G Sepharose (Sigma, St. Louis, MO) as described^{52 (link)}. Test sera were also depleted of IgG to facilitate detection of low levels of IgA antibodies. Macaque IgA was detected using a mixture of biotinylated clone 10F12 (NHP Reagent Resource) and biotinylated clone 40.15.5 (Ward et al 1995) anti-rhesus IgA monoclonal antibodies, which do not cross-react with human IgA and, when combined, appear to recognize all allotypes of rhesus macaque IgA (Kozlowski, personal observation). For RBD IgG assays, the above-pooled rhesus serum standard and a secondary monoclonal antibody specific for macaque IgG were used. Neutralizing assay pseudovirus neutralization assay was performed as described⁵³ .

These assays were done using methods similar to those described^{41 (link)} and Immulon 4 microtiter plates (VWR, Radnor, PA) coated with 100ng per well of S1, S2, N or RBD protein (SinoBiological #40592-VNAH). For IgA assays, a pooled rhesus serum collected day 14 after infection with SARS CoV-2 was used as standard after depletion of IgG using GE Healthcare Protein G Sepharose (Sigma, St. Louis, MO) as described^{41 (link)}. Test sera were also depleted of IgG to facilitate detection of low levels of IgA antibodies. Macaque IgA was detected using a mixture of biotinylated clone 10F12 (NHP Reagent Resource) and biotinylated clone 40.15.5 (Ward et al 1995) anti-rhesus IgA monoclonal antibodies, which do not cross-react with human IgA and, when combined, appear to recognize all allotypes of rhesus macaque IgA (Kozlowski, personal observation). For RBD IgG assays, the above pooled rhesus serum standard and secondary monoclonal antibody specific for macaque IgG were used.