Anti mouse igg1

ELISA-plates were coated with 5x10⁵ plaque-forming units (PFU) heat-inactivated RSV-A2 in 100 μl carbonate buffer (50 mM carbonate/bicarbonate, pH 9.6) per well overnight at 4°C. Subsequently, the binding sites were blocked with 5% skimmed milk in PBS-T (PBS including 0.05% Tween-20) for one hour at room temperature and the plates were washed with PBS-T. Sera, diluted in 2% skimmed milk in PBS-T, were added to the wells. After 1h incubation at 4°C and three washing steps, secondary detection antibodies were added for 1h at RT: HRP-coupled anti-mouse Ig (1:1000, polyclonal, Daco), anti-mouse IgG1 (1:1000, clone X56, BD Biosciences), anti-mouse IgG2a (1:1000, clone R19-15, BD Biosciences), or anti-mouse IgA (1:5000, polyclonal, Bethyl Laboratories). Plates were washed seven times before ECL solution was added and the signal (relative light units per second, RLU/s) acquired on a microplate luminometer (VICTOR X5, PerkinElmer) using PerkinElmer 2030 Manager software.

Antibodies to mouse CD4 (clone GK1.5), CD40 (hamster; clone HM40-3), CD25 (clone PC61.5), lymphocyte activation gene-3 (LAG3; clone eBioC9B7W), programmed cell death protein 1 (PD-1) (clone J43), glucocorticoid-induced TNFR-related protein (GITR; clone DTA-1), inducible T-cell costimulator (ICOS; clone 7E.17G9), cytotoxic T lymphocyte-associated protein 4 (CTLA-4; clone UC10-4B9), forkhead box P3 (FoxP3; clone FJK-16s), MHC II (clone M5/114.15.2), CD80 (clone 16-10A1), CD86 (clone GL1), OX40L (clone RM134L), CD40L (clone RM134L), ICOS ligand (ICOS L; clone HK5.3), PD-1 ligand 1 (PD-L1; clone MIH5), PD-1 ligand 2 (PD-L2; clone122) and isotype control antibodies, including the hamster IgG control (cat. No, 14-4888-81) for the anti-CD40 antibodies, were purchased from eBioscience (San Diego, CA). Anti-mouse IgG1 and IgE were from BD Biosciences (San Jose, CA). All paramagnetic sorting beads and columns were from Miltenyi Biotec (Auburn, CA). Escherichia coli 0127:B8 LPS and OVA were purchased from Sigma-Aldrich (Oakville, ON). GM-CSF and IL-10 were purchased from R&D Systems (Minneapolis, MN).

Mouse anti-β-LG serum was diluted ten-fold in PBS-HS. For the depletion of IgG, diluted antiserum samples were then mixed with an equal amount of Protein G Sepharose 4 Fast Flow (GE Healthcare, Uppsala, Sweden) or Sepharose 4B (SIGMA-ALDRICH, Saint Louis, MO, USA), and incubated for 2 h at room temperature on a rotating platform. Following incubation, the antiserum samples were recovered by centrifugation (500×g for 5 min at room temperature). In order to deplete IgG-subclasses, Streptavidin Sepharose High Performance (GE Healthcare) was mixed with double its volume of each of the following biotinylated rat mAbs at a concentration of 0.5 mg/mL. IgG-subclass specific antibodies (BD Biosciences); anti-mouse IgG1 (clone A85-1, IgG1, κ), anti-mouse IgG2a (clone R19-15, IgG1, κ), anti-mouse IgG2b (clone R12-3, IgG2a, κ), and anti-mouse IgG3 (clone R40-82, IgG2a, κ), and isotype controls (BioLegend) for IgG1 (clone RTK2071) and IgG2a (clone RTK2758). The antibody-bound beads were subsequently washed five times with PBS-HS. Incubation with mouse anti-β-LG serum followed by recovery of the serum samples was carried out as described above. The depletion of IgG and IgG-subclasses from the antiserum was confirmed by ELISA.