Kpl sureblue

To measure IFN autoantibodies in BALF and plasma, ELISA plates were coated with 1 µg/mL IFNα (130-093-874, Miltenyi Biotec) or IFNω (BMS304, Invitrogen) overnight at 4°C, followed by blocking with 5% skimmed milk. Plasma samples were diluted 50× in HPE buffer (M1940, Sanquin) before incubation. Bound autoantibodies were detected with HRP-conjugated goat anti-human IgG, IgA, IgM (GAHu/Ig/Fc/PO, Nordic-MUbio), and HRP substrate, SureBlue KPL (5120-0077, Sera care). BAL samples from patients with CARDS were diluted 3× in 0.4% Triton-X-100 and left for 30 min to ensure inactivation of potential SARS-CoV-2 present in the samples before incubation on the plates. A cut-off of 0.5 (blank-corrected OD450-OD630) was used to determine positivity in plasma samples based on data from previous studies (15 (link)). The cutoff value for determining positivity in BAL samples is unknown.

IFN autoantibodies were measured in serum as previously described (19 (link)). Briefly, ELISA plates were coated with 1 µg/mL IFNα (130-093-874, Miltenyi Biotec) or IFNω (BMS304, Invitrogen) overnight at 4°C followed by blocking in 5% skimmed milk. Serum samples were diluted 50x in HPE buffer (M1940, Sanquin) before incubation. Bound autoantibodies were detected with HRP-conjugated goat anti-human IgG, IgA, IgM (GAHu/Ig(Fc/PO, Nordic-MUbio) and HRP substrate, SureBlue KPL (5120-0077, Sera care).

HlgC quantification was performed by a sandwich ELISA using a custom-made mouse monoclonal antibody (R&D Biotech) and a custom-made polyclonal rabbit F(ab)′₂ biotinylated antibody (R&D Biotech), both raised against HlgC. A 96-well Nunc MaxiSorp plate (Thermo Scientific) was coated with anti-HlgC monoclonal antibody at 10 μg/mL overnight at 20°C. After 5 consecutive washes with phosphate-buffered saline (PBS)–0.05% Tween (PBS-T), wells were saturated for 1 h 30 min at 20°C with a blocking solution containing PBS-T, low-fat milk (5 g/L), and bovine serum albumin (BSA) (1 g/L). Standard dilutions (15 to 1,000 ng/mL) of recombinant HlgC or the culture supernatant were denatured for 1 h at 95°C, loaded in duplicate, and incubated for 2 h at 37°C. The plate was washed, polyclonal rabbit F(ab)′₂ biotinylated antibody (1.55 μg/mL) was added, and the plate was incubated for 1 h 30 s at 37°C. After the incubation, the plate was washed, and ExtrAvidin-peroxidase antibody (Sigma) targeting the biotin molecule and conjugated with horseradish peroxidase (HRP) was added, and the plate was incubated for 1 h at 20°C. A final wash was performed before 75 μL of the substrate tetramethylbenzidine (KPL SureBlue; SeraCare) were added. The reaction was stopped with sulfuric acid at 1 N. The plates were read at 450 nm in a Bio-Rad model 680 microplate reader.