Neutravidin pe

Levels of VRC01 were measured as described in [27 (link)]. Levels of rhesus Rh-α₄β₇ antibody in macaque plasma were measured using the α₄β₇-expressing human T cell line HuT-78 (NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HuT 78 from Dr. Robert Gallo) in a flow cytometry-based assay as described in [16 (link), 22 (link)] using the standard curve method. Briefly, HuT 78 cells were first incubated for 2–3 days in complete RPMI 1640 media containing 100 nM retinoic acid to increase the surface expression of α₄β₇. Cells (150,000/condition) were stained with LIVE/DEAD Aqua dye (Thermo Fisher Scientific, Waltham, MA) for live/dead discrimination, incubated for 30min at 4°C with the plasma to be tested (1:10 diluted in PBS) obtained from macaques from the VRC01-α₄β₇ treatment group before (baseline, BL) and up to 6 weeks after treatment. Cells were then washed and incubated for 30 min at 4°C with anti-rhesus IgG1 (NHP Resource Center, antibody 7H11, in house biotinylated with EZ-link NHS-biotin (Thermo Fisher Scientific) following the manufacturer’s instructions), washed again and resuspended in neutravidin-PE (Thermo Fisher Scientific) for 20 min at 4°C. PE fluorescence was analyzed on a flow cytometer. For the standard curve, baseline plasma was pooled and spiked with serial dilutions of Rh-α₄β₇ (2,500 μg/ml– 0 μg/ml).

Biotinylated recombinant laminin variants Lam521, 511, 421 and 332 and EHS‐laminin111 (Biolamina, Sundbyberg, Sweden) were used in bead‐based laminin‐binding assay. 2 × 10¹⁰ EVs were added to 4 × 10³ CD9 Exosome Capture Beads (Immunostep) in a total volume of 80 μl 3% BSA‐PBS for 3 h at RT. Beads were collected with centrifugation at 3000 × g for 5 min at RT and washed once with 100 μl PBS. Five μg/ml of biotinylated laminin variants in 3% BSA‐PBS were added to the EV‐bead mixture and incubated for 30 min at RT. After pelleting and a washing step with 3% BSA‐PBS at 4°C, neutravidin‐PE (Thermo Fisher Scientific) (1:800) in 3% BSA‐PBS was added for 30 min on ice to detect EV‐bound laminin in a flow cytometry measurement performed on CytoFLEX S instrument (Beckman Coulter). Median fluorescence values of FSC/SSC gated population were expressed as multiple of readings obtained with wild‐type CD81 EVs after values obtained with the secondary reagent alone were subtracted.

Five times 10⁹ recombinant EVs harbouring wild‐type CD81‐eGFP or its laminin‐targeting variant L2ALU_1 were incubated with 6 × 10³ CD9 Exosome Capture Beads (Immunostep) in 120 μl 3% BSA‐PBS for 3 h at RT. Beads were collected with centrifugation at 3000 × g for 5 min at RT and washed once with 100 μl PBS. Five μg/ml of biotinylated human placental laminin in 3% BSA‐PBS, and additionally 15 μg/ml of unlabelled laminin for competition in parallel samples were added to the EV‐bead mixture and incubated for 30 min at RT. After pelleting and a washing step with 3% BSA‐PBS at 4°C, neutravidin‐PE (Thermo Fisher Scientific) (1:800) in 3% BSA‐PBS was added for 30 min on ice to detect EV‐bound laminin in a flow cytometry measurement performed on CytoFLEX S instrument (Beckman Coulter).