Sheath fluid

Maternal plasma samples were analyzed for target biomarkers, namely cytokines [interleukins (ILs), tumor necrosis factor-alpha (TNF-α), interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GMCSF)], chemokines [monocyte chemoattractant protein-1(MCP-1), macrophage inflammatory protein-1 beta (MIP-1β)], vascular endothelial growth factor (VEGF-A), acute phase proteins C-reactive protein (CRP), soluble intracellular adhesion molecule (ICAM-1), soluble vascular cell adhesion molecule (VCAM-1) and matrix metalloproteinases (MMPs) by affinity-based multiplex protein array assays using Bio-Plex Pro Human panels (Bio-Rad, Canada) and Milliplex Map kits (Millipore, Canada). Briefly, plasma samples were incubated with capture antibody-coated magnetic beads, then washed and reacted with biotinylated-detection antibodies followed by incubation with streptavidin-phycoerythrin. The bead complex was washed and re-suspended in sheath fluid (Bio-Rad, Canada) and analysed using a Bioplex 100 instrument with Bioplex Manager 6.0 software (Bio-Rad, Canada).

We adapted the Luminex assay developed by C. Fenwick and coll. to detect anti-HIV-1 gp140z Env-specific IgG [23 (link)]. The in-house produced HIV-1 Env Gp140z trimer was associated with MagPlex beads using the manufacture’s protocol (Bio-Rad, France). Activated beads were washed in PBS followed by the addition of 6.3 μg of protein antigen (4°C overnight under agitation). Beads were then washed with PBS, resuspended in blocking buffer and finally in 150μl of storage buffer. Beads were counted with an Auto-2000 cell counter (Nexcelom) and kept protected from light at 4°C. Luminex beads were diluted at 50,000 beads/mL in PBS and 50μl was added in a Bio-Plex Pro 96-well Flat Bottom Plates (Bio-Rad). Following two 0.05% tween PBS on a magnetic plate washer (Bio-Rad), 50 μl of individual serum samples diluted at 1/100 in PBS, were added. Binding was performed at RT for 30 min under agitation, before adding an anti-mouse IgG-PE secondary antibody (45min at 0.5 μg/mL, ThermoFisher). Beads resuspended in 80 μl of Sheath fluid (Bio-Rad) were agitated 5 min at 700 rpm on the plate shaker then read directly on a Bioplex-200 plate reader (Bio-Rad) with 50μl of acquisition volume and DD gate 5,000–25,000 settings. Median fluorescence intensities (MFI) were exported using Bioplex Manager 6.1 software.