Luminex Assay for Anti-HIV-1 gp140 IgG

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.