Duoset elisa kits for each protein

Soluble protein levels were all measured using DuoSet ELISA kits for each protein (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. DuoSet ELISA ancillary reagent kit (R&D Systems) was used for respiratory fluids and in-house reagents with the same composition were used for plasma samples. In brief, 96-well R&D ELISA microplates (respiratory fluids) or 96-well Nunc Maxisorp ELISA plates (ThermoFisher, plasma) were coated with capture antibody overnight, followed by blocking with 1% w/v BSA for a minimum of 1 h. Samples and standard proteins were added and incubated for 2 h at room temperature, followed by detection antibody for a further 2 h. Lastly, streptavidin-HRP, substrate solution and stop solution (2 N H₂SO₄) were added subsequently for 20 min each. Plates were read on a Multiskan plate reader (Labsystems) using the Thermo Ascent Software for Multiskan v2.4. Plasma samples were diluted in 1:300 for sIL-6Rα ELISAs. Respiratory fluids were diluted in 1:50/1:150 for sIL-6Rα ELISA accounting in the original dilution factors and tested without further dilution for ADAMTS4 ELISA.

Soluble protein levels were all measured using DuoSet ELISA kits for each protein (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. DuoSet ELISA ancillary reagent kit (R&D Systems) was used for respiratory fluids and in-house reagents with the same composition were used for plasma samples. In brief, 96-well R&D ELISA microplates (respiratory fluids) or 96-well Nunc Maxisorp ELISA plates (ThermoFisher, plasma) were coated with capture antibody overnight, followed by blocking with 1% w/v BSA for a minimum of 1 hour. Samples and standard proteins were added and incubated for 2 hours at room temperature, followed by detection antibody for a further 2 hours. Lastly, streptavidin-HRP, substrate solution and stop solution (2N H₂SO₄) were added subsequently for 20 minutes each. Plasma samples were diluted in 1:300 for sIL-6Rα ELISAs. Respiratory fluids were diluted in 1:50/1:150 for sIL-6Rα ELISA accounting in the original dilution factors and tested without further dilution for ADAMTS4 ELISA.