Nr 52286

Genomic RNA for SARS-Related Coronavirus 2 (Isolate USA-WA1/2020), NR-52285, was obtained from BEI Resources. These genomic RNA vials were stored at −80°C and stock volumes were either used directly for experimentation or diluted to the correct concentration in TE Buffer. For experiments using virus, Heat Inactivated SARS-Related Coronavirus 2, NR-52286, was obtained through BEI resources. These stocks were aliquoted and stored at −80°C. Either stock volumes were used for direct experimentation or diluted in TE Buffer or Viral Transport Media to the correct concentrations.

Human serum antibodies IgA (I4036), IgG (I4506) and IgM (I8260) (Sigma-Aldrich, St. Louis, MO) were reduced with 1 M DTT for 10 min to a final concentration of 3 pmol of each antibody on the plate. Human salivary α-amylase (A1031, Sigma-Aldrich, St. Louis, MO) was prepared at 100 pmol/μl in LC-MS grade H₂O, 1 μL of which was spotted. Preparation of positive and negative controls for MALDI-ToF analysis followed closely to that of gargle samples. The negative control, consisting of pooled human saliva (pre-COVID-19) collected before November 2019 (Lee Biosolutions, Maryland Heights, MO), was prepared by spiking 500 µL of the thawed stock into 10 mL of water in a 50 mL tube. From here, the control was filtered and processed following the gargle sample procedure. The positive control (heat inactivated SARS-CoV-2) was obtained from BEI Resources (NR-52286). Briefly, this standard was a heat inactivated, clarified, and diluted cell lysate and supernatant from Vero E6 cells infected with SARS-CoV-2. This sample (225 µL) was treated with 4X v/v of chilled acetone (900 µL) and incubated overnight at −20 °C followed by centrifugation at 10,000 × g for 15 min at 4 °C. The pellet was completely dissolved in 25 µL of the reconstitution buffer.