Luminex

The levels of IgG, IgM, IgA, sIgA, IgG1, IgG2a, IgG2b, and IgG3 in serum and the supernatant of stomach contents, and sIgA from the supernatants of feces and intestinal contents were detected using enzyme-linked immunosorbent assay (ELISA) with commercial kits (Elabscience, Wuhan, PR. China). Aliquots of 10 mg of stomach contents were added to 90 μL of sterile PBS and centrifuged for 3 min at 6000 g to obtain supernatants. Feces (50 mg) were mixed with 200 μL of sterile PBS, vortexed, and then centrifuged for 3 min at 6000 g to obtain supernatants. Serum IgE was determined via ELISA using mouse IgE ELISA kits (Invitrogen, Thermo Fisher Scientific, Inc., MA, USA). Serum OVA-specific IgE and IgG1 were detected using Anti-Ovalbumin IgE (mouse) ELISA kits and IgG1 (mouse) ELISA kits (Cayman Chemical, MI, USA). The serum cytokines IL-1β, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17A, and IFN-γ were measured using Luminex (R&D Systems Inc., MN, USA). Assays were performed according to the manufacturer’s instructions and read using a Multiskan™ GO microplate spectrophotometer (Thermo Fisher Scientific, Inc., MA, USA) and a Luminex 200™ multiplexing instrument (Merck Millipore, MA, USA).

SEBC samples were analysed with a multiplex immunoassay (Luminex, Austin, TX, R&D Systems Cytokines) according to manufacturer's instruction protocol (R&D Systems). The concentrations of a set of cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, IFN-γ, G-CSF, and TNF-α) and chemokines (MCP-1 and MIP-1β) were measured.

EDTA-anticoagulated blood collected at study enrollment (within 72 h of hospital admission) was centrifuged at an ambient temperature at 1000 g for 10 min, and plasma was collected and stored at − 80 °C. SARS-CoV-2 viral nucleocapsid protein (N-antigen) levels were measured in plasma using a single-molecule immune bead assay (Quanterix, Billerica, MA, USA). N-antigen concentrations below the lower limit of detection of 3 pg/mL were assigned a value of 2.9 pg/mL. Plasma interleukin (IL)-6, IL-8, IL-18, IL-10, interferon-gamma induced protein (IP)-10, surfactant protein D (SPD), receptor for advanced glycation end-products (RAGE), angiopoietin-2 (Ang-2), and soluble tumor necrosis factor receptor (sTNFR)-1 were measured using multiplex magnetic bead immunoassays (Luminex, R&D Systems, Minneapolis, MN, USA), and Protein C was measured by ELISA (Helena Laboratories, Beaumont, TX).