Bio plex protm human cytokine assays

Venous blood was obtained after a 12-h fast to evaluate biochemical parameters at the first and sixth visits of the study. Plasma samples were stored at −80 °C until analysis. Plasma inflammatory cytokines were quantified using Bio-Plex ProTM Human Cytokine Assays (Bio-Rad, Hercules, CA, USA). The Bio-Plex Suspension Array System, which was based on Luminex xMAP technology, was a multiplex flow cytometric based system that utilized up to 100 color-coded 8 µm magnetic bead sets. Each bead set was internally dyed with different ratios of two spectrally distinct fluorophores. Each of the bead sets could be conjugated with a unique antibody, antigen, enzyme, and substrates. The conjugated beads were pooled together in the wells of a microplate with the sample to be tested, followed by the addition of a detection antibody, forming a capture immunoassay that was read by the Bio-Plex suspension array reader. Each separate reaction was identified and quantified based on the bead color.

To evaluate the potential role of cytokines, we performed a multipanel assessment of pro-inflammatory cytokines, using a Bio-PlexProTM Human Cytokine Assays (BioRad, Hercules, CA, US CAT.#M500KCAF0Y): interferon (IFN)-γ, interleukin (IL)-1β, IL-2, IL-6, IL-12, IL-17, tumor necrosis factor (TNF)-α were dosed. These studies were performed on 247/324 subjects who agreed to undergo peripheral venous blood sampling at the 12-months follow-up visit; for 73 patients, a blood sample, obtained during the acute phase of disease was also available and was therefore used to assess the potential predictive value of baseline cytokines levels on long COVID development.
Bio-PlexProTM Assays are sandwich immunoassays formatted on magnetic beads. Primitive antibodies directed against the desired biomarker are covalently coupled to the beads. Coupled beads react with the sample containing the biomarker of interest. After a series of washes to remove unbound protein, a biotinylated detection secondary antibody is added to create a sandwich complex. The final detection complex is formed with the addition of streptavidin-phycoerythrin (SA-PE) conjugate. Phycoerythrin serves as a fluorescent indicator, or reporter.
The samples were processed following the steps of the Bio-PlexProTM Assays manual issued by the supplier.