Magpix system

Concentrations of pro-inflammatory cytokines were measured in plasma samples collected at baseline and at the end of the intervention. Paired samples were blinded and analysed in the same batch in the Department of Experimental and Clinical Medicine laboratory (University of Florence, Florence, Italy). Samples were tested for IL-1α, IL-1β, IL-6, and TNF-α levels by MILLIPLEX MAP kit (EMD Millipore, Massachusetts, USA) and for CRP level by Affymetrix ProcartaPlex Multiplex Immunoassays (Invitrogen, California, USA), based on Luminex xMAP technology, according to the manufacturer’s instructions. The upper and lower limits of quantification (ULOQ-LLOQ) for each tested analyte were as follows: 10,000–9.4 pg/ml for IL-1α, 10,000–0.8 pg/ml for IL-1β, 10,000–0.9 pg/ml for IL-6, 10,000–0.7 pg/ml for TNF-α and 9–0.02 mg/L for CRP. Cytokines were detected using the fluorescent imager MAGPIX System (Luminex, Texas, USA) and data were acquired and analysed by xPONENT 4.2 software. The concentration of the samples was calculated by plotting the expected concentration of the standards against the mean fluorescence intensity generated by each standard. A four-parameter logistic algorithm was used for the best curve fit.

MST product functionality was measured with the Bioplex Pro Human 17-plex Cytokine Assay kit (Biorad, Hercules, CA, USA). On Day 1, MSTs from healthy donors were rested overnight with low dose IL-2 (50 U/mL). On Day 2, T cells were washed and plated at 1 × 10⁶ cells/well with 1 μl of corresponding pepmix. Conditions were as follows: No pepmix (control), actin only (control), SEB (positive control), AG85B, PPE68, ESXA, ESXB, or ADK at 1 ug/well. On Day 3, supernatants were harvested from the wells, spun down to remove debris, and plated on the multiplex plate. For immunodeficient patients, supernatants were collected from ELISPOT plates to be run on 17-plex, due to limited cell numbers. The Biorad 17-plex multiplex manufacturer's protocol was followed and read on a MAGPIX System (Luminex, Austin, TX).

Adiponectin and resistin concentrations were analyzed on the Magpix system using the Luminex xMAP technology (Luminex Corporation, Austin, TX, USA). Serum concentrations of adiponectin and resistin were determined simultaneously by the human adipokine 2-plex magnetic bead panel from EMD Millipore Corporation (HADK1MAG-61K-2, Billerica, MA, USA). According to the manufacturer, there was no or negligible cross-reactivity between the antibodies for an analyte and any of the other analytes in this panel. The minimal detectable concentrations were 11 pg/mL for adiponectin and 2.2 pg/mL for resistin. The coefficient variations within and between assays was 4.4% and 10.1% for adiponectin and 5.5% and 11.3% for resistin. All analyses were performed according to the manufactures’ protocols and done in duplicate. The concentrations were calculated from best-fit standard curves generated from calibrators for each analyte in each assay.

RBD proteins from SARS-CoV-2, SARS-CoV, RaTG13, GX-P5L, RmYN02, SL-ZC45, and RacCS203 were all conjugated onto MagPlex-c microsphere (Luminex) using xMAP Antibody Coupling Kit (Luminex) according to the manufacturer’s instructions. To assess ACE2 binding reactivity, 1250 beads/antigen were pre-incubated with PE-conjugated ACE2 (Genscript) for 1 h at 37 °C. The level of ACE2 binding was determined using Luminex MAGPIX system.