Igg3 antibodies

Resting splenic B cells were collected by negative selection with anti-CD43 and anti-CD11b magnetic beads (Miltenyi Biotec) and cultured in RPMI media (Invitrogen) containing 10% (v/v) fetal bovine serum (Sigma-Aldrich), 100 U/ml penicillin-streptomycin (Invitrogen), 2 mM glutamine (Invitrogen), and 50 μM β-mercaptoethanol (Sigma-Aldrich). Cells were plated at 0.5 × 10⁶ cells/ml in 24-well plates and stimulated with 5 μg/ml LPS (E. coli serotype 0111:B4; Sigma-Aldrich) to induce IgG3; LPS plus 5 ng/ml recombinant IL4 (Biolegend) for IgG1; LPS plus 2 ng/ml TGF-β (R&D Systems) for IgG2b; and LPS plus 25 ng/ml IFN-γ (R&D Systems) for IgG2c. Flow cytometry analysis of switched populations was conducted after 4 days using cells stained with FITC- or PerCP-labeled anti-B220 (clone RA3-6B2, eBioscience), and either allophycocyanin-conjugated anti-IgG1 (clone M1-14D12, eBioscience), recombinant PE-conjugated IgG2b, IgG2c, or IgG3 antibodies (Southern Biotech).

Autoantibody titers, total serum antibody levels, and serum autoantibodies by microarray, were performed as described (13 ). Briefly, 96 well Nunc-Immuno MaxiSorp plates (Thermo Fisher) were pre-coated with: Autoantibody ELISAs: calf thymus dsDNA (100ug/ml; Sigma-Aldrich D3664-5X2MG) or Sm/RNP (5ug/ml; Arotec Diagnostic Limited ATR01-10); Total serum antibodies: goat anti-mouse IgM-, IgG-, IgG2c, IgG2b, IgA, IgG1 or IgG3 antibodies (1:500 dilution, SouthernBiotech). Plates were blocked with 1% BSA prior to addition of dilute serum. Specific antibodies were detected using goat anti-mouse IgM-, IgG-, IgG2c, IgG2b, IgA, IgG1 or IgG3-HRP (1:2000 dilution; SouthernBiotech) and peroxidase reactions quantified using OptEIA TMB substrate (BD Biosciences). Autoantigen microarrays were performed at the UT Southwestern Medical Center Microarry Core Facility, Dallas, TX (29 (link)).