Bio plex manager 5

Concentrations of IL-2, IL-4, IL-5, IL-10, IL-13, IL-17, TNF-α, and IFN-γ were measured in pooled cell culture supernatants using a human cytokine kit (Merck-Millipore, Burlington, MA, USA) and multiplex technology according to instructions of the manufacturer. Samples were measured and data were analyzed with Bio-Plex200 and Bio-Plex Manager 5.0 software (Bio-Rad Laboratories).

Bio-Plex Pro^TM Mouse Cytokine GII 9-Plex and Bio-Plex Pro^TM TGF-β 3-Plex assays were purchased from Bio-Rad (Hercules, CA, USA). Supernatants of tumour cell-platelet reactants were analysed according to the manufacturer’s protocol. Signal detection by measurement of fluorescence intensity in each well and data analysis were performed using the Bio-Plex 200 System and Bio-Plex Manager 5.0 (Bio-Rad).

A Milliplex MAP 32-analyte kit for the analysis of mouse cytokines/chemokines (EMD Millipore) was used for measuring 32 cytokines and chemokines simultaneously in each sample. The MAP system is based on unique populations of 100 different, individually identifiable microbead sets coupled to an antibody specific to a cytokine or chemokine that captures the relevant analyte in the sample prior to detection using a second, biotinylated antibody specific to each respective analyte [30 (link)]. Multiplex assays were performed according to the manufacturer’s instructions. Data for each of the analytes were collected as median fluorescent intensity (MFI) that was used to calculate analyte concentrations in pg/mL using the software package BioPlex Manager 5.0 (Bio-Rad) as previously described [31 (link)].

Magnetic carboxylated fluorescently labeled microbeads (Magplex beads, Luminex Corp., Austin, TX, USA) were coupled with Ebola-GP (EBOV rGPdTM by IBT Bioservices). The coupled beads were utilized in a multivariate Luminex assay for antibody class determination. Briefly, beads were diluted to a concentration of 50 beads/μL in Assay Buffer (PBS-1×, 0.1% BSA). Using a black, clear bottom 96-well plate (Greiner Bio One, Kremsmünster, Austria Cat. No. 655906), 50 μL of the working microsphere mixture (2500 beads/well), 40 μL of Assay Buffer and 10 μL of plasma sample diluted 1:10 (1 µL of plasma) were added to each well. The plate was covered and incubated overnight at 4 °C. A Bio-plex array reader (Bio-Plex 200, Bio-Plex Manager 5.0, Bio-Rad Laboratories, Inc., Hercules, CA, USA) detected the microspheres, and binding of PE detector antibody was measured to calculate a Median Fluorescence Intensity (MFI). Background signal, defined as the average MFI observed for each microsphere set when incubated with the PE detector antibody in the absence of a clinical antibody sample, was subtracted from the MFI for each sample. The human anti-EBOV-GP antibody KZ52 (IBT Bioservices Cat. No. 0260-001) was used as a positive control.

The Corona Array is an in-house bead-based 10-plex suspension array based on the xMAP^® technology (Luminex^®, Austin, Texas, USA) for the simultaneous analysis of antibodies of different specificities in one sample. The 10-plex included 6 recombinant His-tagged proteins/protein subunits of SARS-CoV-2 and 4 recombinant S1 subunits of HCoVs (Supplementary Table 2). The proteins were covalently coupled to MagPlex magnetic microspheres at 100 µg per 1.25 × 10⁷ beads, and the coupling efficiency was determined as previously described in detail (32 (link)).
The Corona Array was performed with plasma as well as MENSA and MENSA+ samples as previously described (32 (link)). Briefly, different seven-step dilution series were prepared in bead buffer based on the expected range of signals: (i) 1:20–1:312,500 (plasma); (ii) 1:1–1:64 (MENSA); and (iii) 1:1–1:729 (MENSA+). A plasma pool (prepared from plasma samples of all AICOVI donors on day 14 after the second vaccination) was included on each plate for data normalization. Antibody binding was determined on the BioPlex 200 system (Bio-Rad Laboratories GmbH; Feldkirchen, Germany), with bead buffer serving as blank, using the following instrument settings: bead type: MagPlex beads; beads: 100 beads per region; sample timeout: 60 sec; sample volume: 80 μL; gate settings: 7,500–15,000 (BioPlex Manager 5.0; Bio-Rad Laboratories GmbH).

PBdMC secreted products were quantified using ProcartaPlex Mix&Match 22‐plex (ebioscience) according to the manufacturer's instruction. The assay measured; Eotaxin (49), Eotaxin‐3 (0.7), GM‐CSF (13), IL‐1beta (2.0), IL‐4 (43), IL‐5 (16), IL‐8 (8.5), IL‐10 (2.2), IL‐13 (8.5), IL‐18 (25), IL‐22 (27), IL‐23 (13), IL‐31 (55), IL‐33 (2.1), IL‐37 (15), MCP‐1 (4.7), MIP‐1α (0.8), RANTES (1.1), TNF‐α (25), TSLP (2.2), VEGF‐A (20), and VEGF‐D (1.6), numbers in parenthesis designate detection limit (pg/ml). The samples were analyzed on a Bio‐Plex 200 system using Bio‐Plex Manager 5.0 (BioRad, Berkeley, CA).