Anti-nucleosome ELISAs were performed by coating Immulon 2HB plates with 10 μg/ml poly-L-lysine (Sigma-Aldrich) in PBS. Plates were washed and coated with 15 μg/ml dsDNA prepared by digestion of calf thymus DNA (Sigma-Aldrich) with S1 nuclease (Promega) for 30 min at 37 deg followed by ethanol precipitation. Plates were subsequently washed and coated with 10 μg/ml calf thymus histones type IIAS (Sigma-Aldrich). Plates were blocked with ELISA buffer (1× PBS 1% BSA 0.05% sodium azide) and serum samples diluted 1:200 in the same buffer were applied to the top row and diluted threefold down the plate down to 1:5,400. Bound antibody was detected with alkaline-phosphatase conjugated goat anti-mouse IgG (Southern Biotech) or goat anti-mouse IgG2a (Southern Biotech) and developed with pNPP (Sigma-Aldrich). Autoantibody concentrations were determined relative to PL2-3 anti-nucleosome monoclonal antibody standard using DeltaSoft 2.8.11 software (Biometallics). Anti-RNA ELISAs were performed in a similar fashion, except plates were coated first with poly-L-lysine then with 15 μg/ml total yeast RNA (Sigma-Aldrich) before blocking and concentrations were determined relative to BWR4 standard. Total IgG and IgM ELISAs were performed by coating plates with unconjugated goat anti-mouse IgG or IgM antibody (Southern Biotech), followed by serum sample diluted 1:10,000 (IgM) or 1:50,000 (IgG) in the top row, followed by goat anti-mouse IgG-AP or IgM-AP (Southern Biotech), relative to purified IgG or IgM standards.