Bio plex manager software version 6

Assay proteins were coupled to a predetermined number of carboxylated magnetic microsphere bead sets, MagPlex® (Luminex Corporation, Austin, TX, USA). Each of the eight protein‐coupled microsphere bead sets were mixed for the multiplex assay and added to individual wells of a 96‐well plate. Serum or plasma samples were heat treated at 56°C for 30 min to inactivate complement, diluted 1:50 in PBSA and 100 μl was added to wells with the microsphere bead sets and incubated for 30 min. Liquid was removed and biotinylated Protein A (1:500) (Pierce, Thermo Fisher Scientific, Waltham, MA, USA) and biotinylated Protein G (1:250) (Pierce, Thermo Fisher Scientific, Waltham, MA, USA) were added to wells and incubated for 30 min at room temperature. Liquid was removed and 100 μl of streptavidin‐phycoerythrin (1:1000) (Qiagen Pty Ltd, Australia) was added and incubated for 30 min at room temperature. After incubation, samples were analysed for antigen‐bound IgG, expressed as a median florescence intensity (MFI) using a Bio‐Plex 200 HTF multiplexing system with Bio‐Plex Manager Software version 6.2 (Bio‐Rad Laboratories, Hercules, CA, USA), with settings set for 100 beads per region and calibrated on the high RP1 target setting.

Plasma samples were analyzed for IL-1β, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, soluble CD40 ligand (sCD40L), IFN-γ, and TNF-α using a 15-plex Human IL-17 Cytokine Kit (Bio-Rad, Hercules, CA, USA). All assays were performed according to the instructions provided by the manufacturer. Briefly, median fluorescence intensities were collected on a Luminex-200 instrument (Bio-Rad) using Bio-Plex Manager software version 6.2 (Bio-Rad). Standard curves for each cytokine were generated using the premixed lyophilized standards provided in the kits, and cytokine concentrations were determined from the standard curve using a 5-point regression to transform the median fluorescence intensity values into concentrations. Each sample was run in duplicate, and the average of the duplicates was used as the measured concentration. Any value that was below detection level was replaced by the limit of detection (LOD) as reported by Luminex kit. Analyses were performed using Data Pro Manager 1.02 (Bio-Rad) and GraphPad Prism Software version 5.0c (La Jolla, CA, USA).