Human ifn ω recombinant protein

Plasma samples from the severe COVID-19 case and the 5 asymptomatic cases were isolated from blood via standard centrifugation. Elisa was performed as described previously [35 ]. Briefly, 96-well ELISA plates (Nunc-Immuno™ MicroWell™ 96 well solid plates; Thermo Fisher Scientific, MA, USA; catalog no. 167008) were coated by incubation overnight at 4 °C with human IFN-α2 recombinant protein (2 μg/ml; Miltenyi Biotec, Bergisch Gladbach, Germany; catalog no. 130-093-874) and human IFN-ω recombinant protein (eBioscience, CA, USA; catalog no. BMS304). Coated plates were then washed (PBS, 0.005 % Tween 20), blocked by incubation with 5 % nonfat milk powder in the same buffer, washed, and incubated with 1:250 dilutions of plasma from the patients or controls for 2 h at room temperature. Horseradish peroxidase (HRP)-labelled anti-human IgG, IgM or IgA (Abcam, Cambridge, MA, USA; catalog no. ab102420) was added to a final concentration of 2 μg/ml. To develop the plate, 100 μl/well of TMB substrate solution (TMB Substrate Kit, Thermo Fisher Scientific, MA, USA; catalog no. 34021) was added and incubated for 5 min. The reaction was stopped with 2 M sulfuric acid (100 μl/well) and optical density at 450/630 nm was measured via BioTek EL808 microplate reader.