HEp-2 immunofluorescence assays (Antibodies, Davis, CA) were performed as previously described (14 (link)) with serum diluted at 1/200 and were scored for relative fluorescence intensity of nuclear and cytoplasmic staining on a scale of 0–3 and for the presence or absence of mitotic chromatin by an observer blinded to the genotype of the mice.
For anti-nucleosome ELISA, polystyrene plates were coated with poly-L-lysine (Sigma-Aldrich, St. Louis, MO). Plates were then incubated with phenol-extracted and S1 nuclease-treated dsDNA from calf thymus (Sigma-Aldrich), followed by calf thymus histones type II-AS (Sigma-Aldrich). After blocking with 1% BSA in PBS, serial dilutions of serum from 1/200 to 1/5400 were added. Specific Abs were detected with alkaline phosphatase-conjugated goat anti-mouse IgG (Southern Bio-technology Associates, Birmingham, AL), and absorbance at 405/630 nm was compared with a purified PL2-3 monoclonal anti-nucleosome standard (27 (link)) for quantitation.
Anti-Sm ELISA was performed as previously described (14 (link)) with serial dilutions of serum from 1/200 to 1/5400. ELISAs for anti-ribosomal P Ag autoantibodies and anti-RNA Abs were described previously (28 (link)).
Anti-IgG2a RF titers were determined by ELISA essentially as described previously (29 (link)). Polystyrene plates were coated with purified mAb 23.3 (IgG2a,λ anti–4-hydroxy-3-nitro-phenylacetyl) overnight. After blocking with 1% BSA in PBS, serial dilutions of serum from 1/200 to 1/5400 were added. Specific Abs were detected with biotinylated anti-κ L chain (clone 187.1; BD Pharmingen, San Diego, CA), followed by alkaline-phosphatase–conjugated streptavidin (Invitrogen, Carlsbad, CA), and absorbance at 405/630 nm was compared with a 400tμ23 (IgM-RF) standard for quantitation.
Total serum IgG was determined by ELISA as described previously (14 (link)). Titers of individual IgG isotypes were measured by cytometric bead assay (Millipore, Bedford, MA) per the manufacturer’s protocols.
Serum IL-6, TNF-α, IL-12p40, IL-12p70, and IL-10 were measured by multiplex cytometric bead assay (Bio-Rad, Hercules, CA) per the manufacturer’s instructions. Serum IL-23(p19/p40) was measured by ELISA (eBioscience, San Diego, CA). Serum IFN-α was measured as previously described (14 (link)) with serum diluted 1/20.
For anti-nucleosome ELISA, polystyrene plates were coated with poly-L-lysine (Sigma-Aldrich, St. Louis, MO). Plates were then incubated with phenol-extracted and S1 nuclease-treated dsDNA from calf thymus (Sigma-Aldrich), followed by calf thymus histones type II-AS (Sigma-Aldrich). After blocking with 1% BSA in PBS, serial dilutions of serum from 1/200 to 1/5400 were added. Specific Abs were detected with alkaline phosphatase-conjugated goat anti-mouse IgG (Southern Bio-technology Associates, Birmingham, AL), and absorbance at 405/630 nm was compared with a purified PL2-3 monoclonal anti-nucleosome standard (27 (link)) for quantitation.
Anti-Sm ELISA was performed as previously described (14 (link)) with serial dilutions of serum from 1/200 to 1/5400. ELISAs for anti-ribosomal P Ag autoantibodies and anti-RNA Abs were described previously (28 (link)).
Anti-IgG2a RF titers were determined by ELISA essentially as described previously (29 (link)). Polystyrene plates were coated with purified mAb 23.3 (IgG2a,λ anti–4-hydroxy-3-nitro-phenylacetyl) overnight. After blocking with 1% BSA in PBS, serial dilutions of serum from 1/200 to 1/5400 were added. Specific Abs were detected with biotinylated anti-κ L chain (clone 187.1; BD Pharmingen, San Diego, CA), followed by alkaline-phosphatase–conjugated streptavidin (Invitrogen, Carlsbad, CA), and absorbance at 405/630 nm was compared with a 400tμ23 (IgM-RF) standard for quantitation.
Total serum IgG was determined by ELISA as described previously (14 (link)). Titers of individual IgG isotypes were measured by cytometric bead assay (Millipore, Bedford, MA) per the manufacturer’s protocols.
Serum IL-6, TNF-α, IL-12p40, IL-12p70, and IL-10 were measured by multiplex cytometric bead assay (Bio-Rad, Hercules, CA) per the manufacturer’s instructions. Serum IL-23(p19/p40) was measured by ELISA (eBioscience, San Diego, CA). Serum IFN-α was measured as previously described (14 (link)) with serum diluted 1/20.
