Magnetic carboxylated fluorescent beads

A luminex assay was used to determine relative IgG titers to the HIV-1 YU2 gp120 antigen, as described previously (Brown et al., 2012 (link)). Briefly, magnetic carboxylated fluorescent beads (Luminex Corporation) were coupled to gp120 in a two-step carbodiimide reaction. Serum samples diluted at 1:100 were then incubated with antigen-coated beads overnight at 4 °C. Following bead washing, gp120-specific IgG1 was then detected with PE-conjugated mouse anti-human IgG1 (Southern Biotech #9052-09), diluted to 1.3 μg/ml in assay buffer, to each well. Median Fluorescence Intensity corresponding to each sample was measured using a BioPlex array reader (BioPlex 3D, Bio-Rad). A minimum of 50 beads per well were collected for analysis.

HIV antigens were coupled to magnetic carboxylated fluorescent beads (Luminex Corporation) as described previously (Brown et al., 2017 (link)). Briefly, a total of 5 million carboxylated beads (400 μl) were covalently coupled to 25 μg of antigen using a two-step carbodiimide reaction, and then blocked and suspended in PBS (Phosphate Buffered Saline) -TBN (PBS-1×, 0.1% BSA, 0.02% Tween 20, 0.05% Sodium Azide, pH 7.4, Teknova). The coupled beads were counted (TC-10 cell counter, BioRad) and stored at −80 °C for up to 6 months or at 4 °C for up to 1 month prior to use. Antigen purity was known for most but not all materials used, and was generally >90% as assessed by HPLC, SDS-PAGE, or Edman sequencing/amino acid analysis/mass spectroscopy. Consistent performance of differing antigen or conjugation batches is ensured by meeting acceptance criteria in bridging experiments.

The ability of antigen-specific Abs to bind to FcgRs was assessed using Luminex technology following published protocols [50 (link)]. In brief, magnetic carboxylated fluorescent beads (Luminex Corp.) of different regions were coupled to biotinylated antigens in a two-step carbodiimide reaction. First, beads were washed in PBS with 0.05% Tween-20 and activated for 30 min in 100mM monobasic sodium phosphate, pH 6.2, with freshly added 5 mg/ml of Sulfo-NHS and EDC (both Pierce). Subsequently, beads were washed in 50 mM MES, pH 5.0, and incubated with antigen (antigen:bead ratio in w/v = 12.5%) for 2h on a rotational mixer. Antigen-coupled beads were washed and blocked for 30 min in PBS-TBN (PBS-1×, 0.1% BSA, 0.02% Tween 20, 0.05% sodium azide, pH 7.4), incubated with serum samples overnight at 4°C under shaking, and washed. To measure FcgR binding, biotinylated FcgR receptors (Duke Human Vaccine Institute) were coupled to streptavidin-PE (Phycolink), quenched in 5mM biotin (Avidity) and used for detection. All assays were measured on a BioFlex 3D (Bio-Rad).