Tmb substrate

A 96-well flat-bottom half area plate (Costar) was coated with 10 μg/ml mBSA in coating buffer (15 mM Na2CO3 and 35 mM NaHCO3) for 1 h and blocked with 2% FCS (PAA Laboratories GmbH) in PBS for 2 h. Serum from AIA mice (day 10) was diluted 1:150 with blocking buffer and incubated for 2 h. HRP conjugated goat anti-Mouse IgG1-antibody (Bethyl) was diluted 1:10.000 in blocking buffer and loaded for further 1 h. The horseradish peroxidase activity was induced by TMB-substrate (Merck), stopped with 6% orthophosphoric acid and the signal was assessed using a Wallac 1420 Victor2 Microplate Reader (Perkin Elmer) at 450 nm. All incubation steps were performed at RT.

Recombinant proteins were constructed as previously described^{28 (link)} (Supplementary Fig. S3). The specific IgG and IgA antibodies against recombinant Rv2659c, Rv1738 and ESAT-6 proteins were investigated using an indirect ELISA. Ninety-six-well microplates (Corning, NY, USA) were coated with 25 µl of Rv2659c, Rv1738 and ESAT-6 proteins at 20 ng/µl in HBS buffer (HBS containing 1 mM MnCl₂) and incubated overnight at 4 °C. After washing, plates were blocked with blocking buffer (5% BSA in HBS pH 7.6) for 2 h at RT. The 25 µl diluted plasma samples (1:200 and 1:100 dilution for IgG and IgA detection, respectively) were added and incubated for 1 h at RT. All plasma samples and controls were run in duplicate on the same plate. After a washing step, plates were incubated for 1 h at RT with 25 µl with one of the following secondary antibodies: pre-adsorbed goat anti-human IgG H + L-horseradish peroxidase (Abcam, UK) or peroxidase-labelled anti-human IgA (KPL, USA). Plates were washed and enzyme activity was detected by adding 25 μl/well TMB substrate (Merck KGaA, Sigma-Aldrich, USA) for 15 min. The reaction was stopped with 25 μl/well 1N HCl and the absorbance was read at OD 450 nm.

Recombinant human DDX42 with an N-terminal GST tag was purchased from NovusBio (cat# H0001325-P01). Streptavidin-Coated High-Binding Capacity 96-well plates (ThermoScientific prod #15501) were hydrated with PBS (30 min) and coated with 50 nM biotinylated oligonucleotides (1 hr, shaking 450 rpm). Wells were washed three times with ELISA buffer (50 mM K₂HPO₄ pH 7.4 and 100 mM KCl/100 mM LiCl); 1 min shaking, 450 rpm. Wells were then blocked with 3% (w/v) BSA (Merck, cat# A7030) in ELISA buffer for 1 hr, at room temperature and then incubated with serial dilutions of DDX42 up to 200 nM for 1 hr. Wells were washed three times with 0.1% TWEEN-20 in ELISA buffer and then incubated for 1 hr with anti-GST HRP-conjugated antibody (Abcam AB3416) diluted 1:10,000 in blocking buffer. Wells were again washed three times with ELISA-Tween, and the bound anti-GST HRP detected with TMB substrate (Merck,cat#T4444) for 2 min. Reactions were stopped with 2 M HCl. Absorbance at 450 nm was measured with PheraSTAR plate reader (BMG labtech). Binding curves with standard error of the mean (SEM) were fitted using GraphPad Prism software, using a non-linear regression fit, one site, specific binding model with saturation kinetics. The following equation was used: y=(Bmax*x)/(K_d +x), where x = concentration of DDX42 (nM) and Bmax is the maximum specific binding (i.e. saturation).

For antigen-mediated ELISA screening, 96-well high-binding plates (Costar) were coated with purified proteins (monomer and polymers of A1AT variants) at 2 μg/mL in PBS (Oxoid), probed with hybridoma culture media with detection by a rabbit anti–mouse HRP (0.2 μg/mL, MilliporeSigma A0545). For sandwich ELISA, plates were coated with a rabbit polyclonal anti-A1AT (DAKO A0012) or mAb_2C1 capture antibody at 2 μg/mL, followed by incubation with the target samples, the primary antibody (1 μg/mL), and subsequent detection by either an anti–mAb-HRP secondary (0.2 μg/mL, MilliporeSigma) or direct detection of an HRP-conjugated primary antibody. The complex was revealed with TMB substrate (MilliporeSigma) according to manufacturer’s instructions and the absorbance at 450 nm measured by a SpectraMax M5 plate reader (Molecular Devices).

Levels of IgG, IgA, and IgM antibodies in human sera and animal sera were measured by an indirect ELISA with recombinant proteins (S, S1, RBD, S2, N, E, and NS3 of SARS-CoV-2 and S, N, E, M, and 3CLpro of SARS-CoV), and whole inactivated virus antigens (SARS-CoV-2, HCoV NL63, CCoV 1-71, and H1N1 influenza virus [A/California/2009/07]), which were prepared by density-gradient purification, followed by γ-irradiation or β-propiolactone inactivation. Heat-inactivated sera (56 °C for 30 min) were added to the wells of antigen-coated plates and then detected by the respective horseradish peroxidase (HRP)-conjugated secondary antibodies (SouthernBiotech, Birmingham, AL, USA). TMB substrate (MilliporeSigma, Burlington, MA, USA) was added to produce a color change. The reaction was stopped with sulfuric acid, and optical density (OD) at 450 nm was read using the Synergy plate reader (Biotek, Broadview, IL, USA). The cutoff values of tests were defined as 3 times the average of the negative control samples for human sera, 3 times the average of the SPF samples for canine and feline sera, and 3 times the average of the preimmune murine sera.

A 96-well plate was coated with rabbit polyclonal anti-citH3 (Abcam catalog ab5103) or anti–neutrophil elastase (Calbiochem catalog 481001) at 2.5 μg/mL in PBS overnight at 4°C. Wells were blocked in blocking buffer (1% BSA in PBS) at room temperature (RT) for 1 hour. Plasma or serum were diluted 1:100 in blocking buffer and incubated overnight at 4°C. After washing 3 times with washing buffer (0.05% Tween in PBS), samples were incubated with mouse monoclonal anti–double stranded DNA Ab (MilliporeSigma, clone BV16-13, MAB030) at 1:100 in blocking buffer for 1 hour at RT. The plate was washed 3 times and incubated with goat anti-mouse conjugated HRP Ab (1:10,000) (Bio-Rad, catalog 1706516) for 1 hour at RT. Wells were washed 5 times with 0.05% Tween in PBS followed by addition of 100 μL of TMB substrate (MilliporeSigma) and 50 μL of 0.16 M sulfuric acid stop solution (MilliporeSigma). Absorbance was measured at 450 nm on an ELISA plate reader (Synergy HT, Bio-Tek). ELISA was performed multiple times to ensure reproducibility after freeze-and-thaw cycles and even at high temperature (60°C) for 30 minutes. Data are provided in Supplemental Table 8.