Igg i4506

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.