Protein g hp multitrap

IgG was purified from plasma samples from healthy individuals, or from HIV positive individuals (14 (link)), via Protein G chromatography (Protein G HP Multitrap, GE Healthcare, Sweden) and Melon Gel chromatography (Melon Gel IgG Purification Kit, Thermo Fisher Scientific, USA). For Protein G sepharose purification, plasma was diluted 2-fold with binding buffer (20 mM sodium phosphate, pH 7.0) and incubated for 1 h. IgG was eluted with 0.1 M glycine-HCl (pH 2.7), into neutralizing buffer (1 M Tris-HCl, pH 9.0). IgG was purified from plasma samples by Melon Gel purification according to the manufacturer's protocol. Briefly, serum samples were diluted 1:10 and the diluted serum was added to a minispin column containing the Melon Gel resin. After 5 min incubation, the purified IgG was collected in the flow through following the manufacturer's instructions. Eluted IgG (Protein G) and flow through (Melon Gel) were concentrated, and buffer exchanged into PBS using a 30 kDa Amicon Ultra centrifugal filter (Millipore, USA). IgG concentrations were quantitated using the Human-IgG ELISA kit (Mabtech, Sweden).

Depletion and purification of IgG antibodies from plasma samples was performed using a Protein G HP MultiTrap column (GE Healthcare, Inc.) as previously described [85 (link)].

ADP was performed by measuring the internalization of opsonized HA-coated beads by a phagocytic cells line (Fig 1A). The methods were similar to those previously described for HIV [19 (link), 20 (link)] with minor modifications. Briefly, FITC-labeled NeutrAvidin^® FluoSpheres^® (beads 1μm, Invitrogen, Carlsband, CA) were labeled both with internalization probe tagged with Cy5 (FIP_Cy5) [21 (link)] and 0.75μg biotinylated HA or HIV-1 gp140 then opsonized with 10μg/ml purified IgG (Protein G HP Multitrap, GE Healthcare, UK). 1x10⁵ THP-1 (ATCC TIB-202) cells were incubated with the beads for 16 hr. A 16hr incubation provided a reasonable balance between ADP and non-specific bead uptake (not shown). Cell surface FIP_Cy5 was quenched with a complimentary probe so that internalized beads (FITC+Cy5+—i.e. truly phagocytosed) can be measured. Cells were fixed and 5x10⁴ cells were analyzed by flow cytometry. Background levels of ADP activity were assessed against HIV-1 gp140 since all donors were HIV-negative and calculated as the mean plus 2 SD. Background ADP levels were reproducible across multiple experiments (11–14%) as illustrated in the dotted lines Figs 1B and 3A–3C.