Purified human igg

Monocytes were first incubated with 200 μg/ml of purified human IgG (Sigma) in PBS supplemented with 2 mM EDTA and 1% FCS for 5 min at 4°C to prevent unspecific binding through Fc receptors. Incubations with specific antibodies were carried out for 15 min at 4°C with ready-to-use concentrations of FITC-conjugated antibodies against CD14 (BD Pharmingen, San Jose, CA, USA) and PE-conjugated antibodies against CD54 (BD Pharmingen) or APC-conjugated antibody against CD11a (BD Pharmingen). Labeled cells were washed with PBS and re-suspended in PBS supplemented with 2 mM EDTA and 0.5% BSA. The data was acquired on a Dako Cyan flow cytometer (Beckman Coulter, Fullerton, CA, USA) and analyzed with the Summit software.

Multi-parametric flow cytometry was used for immunophenotyping. PBMC (1 × 10⁶) were stained with a T-cell panel, an innate lymphoid panel or with an isotype control panel. Before antibody labeling, cells were incubated with purified human IgG (Sigma) to reduce non-specific binding. Cells were stained with mAbs against CD3-PE Cy7 (clone SK7), CD4-BD Horizon v500 (clone RPA-T4), CD8-BD Horizon v450 (clone RPA-T8), CD45R0-PECF594 (clone UCHL1), CD62L-APC (clone DREG-56), CD25-APC Cy7 (clone M-A251), CD127-FITC (clone HIL-7R-M21), CD279-PE (clone EH12.2H7) (Biolegend), HLA-DR-PerCPCy5.5 (clone LN3) (eBioscience), CD16-APC H7 (clone 3G8), CD56-APC (clone NCAM 16.2), iNKT-PE (6B11), Vδ2-FITC (clone B6), IgG1k-APC Cy7 (clone MOPC-21), IgG1k-APC (clone MOPC-21) (Biolegend), IgG1k-FITC (clone MOPC-21) (Biolegend), IgG1k-PE (clone MOPC-21) (Biolegend), and IgG2b-PerCPCy5.5 (clone N/S) (eBioscience). Antibodies were from BD Biosciences unless stated otherwise.

Whey was separated on 12% SDS-PAGE gels under both reducing and non-reducing conditions. Anti-CD20 mAb was detected using an HRP-conjugated goat anti-human Fab-specific antibody (Sigma, St. Louis, MO) and ECL Western blot reagents (GE Healthcare). Purified human IgG (Sigma) was used as a positive control.

Other reagents include purified human IgG (Sigma, St. Louis, MO), goat anti-human IgG (Thermo Scientific, Rockford, IL), and goat anti-human IgG conjugated with fluorescent dye (DyLight649; KPL, Inc.). Assay reagents include bovine serum albumin (BSA; Sigma Life Science, St. Louis, MO), phosphate-buffered saline (PBS), blocker casein in PBS, and Tween 20 (Thermo Scientific, Rockford, IL).

Antibody binding toward the RSV F‐protein, in either preF or postF conformation, was performed by Gyrolab microfluidic immunoassays as previously described (Giuliani et al,2018). All supernatants containing naturally produced IgGs were diluted 1:2 in Rexxip H‐Max (Gyros) and analyzed using Gyrolab Bioaffy 200 CDs (Gyros) and the standard Gyrolab three‐step method (capture–analyte–detection) for qualitative screening. Biotinylated preF and postF (captures) were prepared using the EZ‐Link sulfo‐NHS‐LC‐Biotin (Thermo Scientific) at a molar ratio of 10 biotin : 1 protein and used at 100 µg/ml (diluted in PBS/Tween20 0.01%). Alexa 647‐conjugated Goat Anti‐Human IgG (Jackson Immuresearch) was used as detection reagent and prepared at 25 nM diluted in Rexipp F buffer (Gyros). To quantify IgG within each supernatant, a standard curve was prepared by using purified human IgG (Sigma‐Aldrich) starting at 4 µg/ml and diluted step 1:4 in Rexxip H (Gyros), while samples were diluted 1:4 in Rexxip H (Gyros). Following their expression, mAbs were further screened for quantitative binding to both preF and postF, and capture/detection reagents were prepared as described above. Samples and known neutralizing antibodies (D25, Mota and 101F) were prepared at a starting concentration of 1 µg/ml in dose–response curves to select the range of linearity of the response.

Vero cells (5x10⁵) were plated in 6-well plates in DMEM containing 10% FCS and 1% penicillin streptomycin. The following day, brains were extracted from mice and homogenized in 1ml of medium. Brains were centrifuged for 10 min at 8000 g. Supernatant was collected and serially diluted 2-fold in DMEM. 500ul of each dilution was added to the Vero cells for 1 hour with gentle shaking. After 1 hour, medium was removed and 2 ml DMEM containing 15 μg/ml purified human IgG (Sigma I4506). Cells were incubated at 37°C for 2 days. Medium was then removed, and cells were fixed in 100% ice cold methanol for 3 min. Plaques were stained with 10% crystal violet for 20 min with gentle shaking, washed and counted.