Bio plex manager v5

SARS-CoV-2-specific antibodies were quantified using assays previously described^{16 (link)}. Briefly, a standard ELISA assay (data collected with the Multiskan Spectrum; Thermo Fisher Scientific), using as target antigens the extracellular domain of the spike protein in the form of a trimer (ELISA tri-S), and the S-Flow assay, which is based on the recognition of SARS-CoV-2 spike protein expressed on the surface of 293T cells (293T-S), were used to quantify SARS-CoV-2-specific IgG and IgA subtypes in plasma and nasopharyngeal swab supernatants. Briefly, specific IgG and IgA were detected in S-Flow assay by anti-IgG Alexa Fluor 647 (A-21445, Thermo Fisher Scientific, polyclonal; dilution 1:600) or anti-IgA Alexa Fluor 647 (109-605-011, Jackson ImmunoResearch, polyclonal; dilution 1:800). S-Flow assay is a flow cytometry-based assay. The data were acquired with an AttuneTM NxT v3.2.1243.0 and analyzed with FlowJo v10. Assay characteristics including sensitivity and specificity were previously described^{16 (link)}. Total IgA, IgM, IgG1, IgG2, IgG3 and IgG4 were determined using the Bio-Plex Pro Human Isotyping Assay Panel (Bio-Rad) according to the manufacturer’s instructions. Data were acquired on a Bio-Plex 200 system (Bio-Rad) and analyzed using Bio-Plex Manager v5 (Bio-Rad).

16HBE14o- cells were seeded on a 12-well culture plate at a density of 4×10⁵ cells/well. Two days later, culture medium was replaced with fresh medium containing inactivated-H5N1 (20 μg/ml H5 hemagglutinin). A further thirty-six hours later, the medium was harvested and analyzed for IL-6 and CXCL8 by a magnetic bead-based multiplex quantitative cytokine immunoassay (BioPlex) using a protocol provided by the BioRad. Briefly, antibody–conjugated beads were added to individual wells of a 96-well filter plate and adhered using vacuum filtration. After washing with a washing buffer, 50 μl of prediluted standard or culture medium was added to each well and the plate shaken at 300 rpm for 30 min at room temperature. Thereafter, prediluted multiplex biotin conjugated detection antibody was added, followed by a 30 min incubation on the shaker. After washing, prediluted streptavidin-conjugated PE was added to each well and incubated for 10 min. Wells were washed with washing buffer before the BioPlex assay buffer was added. After 5 min incubation, concentrations of each cytokine were determined using a BioPlex 200 instrument equipped with BioPlex Manager V 5.0 software (BioRad). Each sample was run in duplicate and the mean value reported. Data were normalized against control untreated groups and obtained from at least 3 independent experiments.

Serum samples were obtained from the participants using their fasting blood in the early morning. The following cytokines were quantified using a 27-plex magnetic bead based immunoassay kit (Bio-Rad, Hercules, CA, United States): interleukin-1β (IL-1β), IL-1 receptor antagonist (IL-1ra), IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, eotaxin, fibroblast growth factor-basic (FGF-basic), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-γ), interferon gamma-inducible protein 10 (IP-10), monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), platelet-derived growth factor (PDGF-bb), MIP-1β, regulated upon activation normal T-cell expressed and secreted (RANTES), tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF). The Bio-Plex 200 system (Bio-Rad) was used to analyze Bio-Rad 27-plex human group I cytokines, with the Bio-Plex assay performed according to the manufacturer’s directions. The results were expressed as picograms per milliliter (pg/mL) using standard curves integrated into the assay and the Bio-Plex Manager v5.0 software (Bio-Rad), yielding reproducible intra-and inter-assay CV values of 5–8%.