Calf thymus dsdna

Serum samples were taken from mice at the beginning (eight weeks) and end (15 weeks) of the experiments and were stored at -35°C until anti-dsDNA (15 (link)), and anti-Histone (22 (link)) antibodies were analyzed by ELISA as follows. A MaxiSorp plate (Nunc, Rochester, NY, USA) from 96-well was coated with 2.5 μg/ml calf thymus dsDNA (Sigma Aldrich, St Louis, MO, USA) or 10 μg/ml calf thymus histone (Roche Diagnostic, Mannheim, Germany) in 100 μl of bicarbonate buffer overnight at 4°C. Bovine serum albumin (BSA, Invitrogen, Carlsbad CA, USA) at 2% was used to block the plate. The plate was incubated for one hour at 37°C with serum (1:50) or the anti-dsDNA antibody standard (clone 16-13, Chemicon International, Billerica MA, USA) or was incubated for two hours at room temperature with serum (1:150) for anti-histone antibody. The plate was washed and incubated with rabbit anti-mouse IgM, IgG, IgG1, or IgG2a conjugated to alkaline phosphatase (AP, Zymed Laboratories, San Francisco CA, USA) or anti IgG2b or IgG3 conjugated to peroxidase (HRP), after which substrate was added. (5-bromo-4-chloro-3- indolyl phosphate; Sigma−Aldrich, St Louis MO, USA) for AP or HRP substrate (3.3’,5,5’ tetramethylbenzidine TMB Sigma−Aldrich), respectively. The O.D. was read at 405 or 450 nm using a Dynatech MR5000 ELISA reader.

Direct ELISA assays were performed similarly to those reported previously. Briefly, high-binding Costar 96-well plates (Corning) were coated with 0.5 μg salmon sperm ssDNA (Abcam), calf thymus dsDNA (Sigma-Aldrich), lipopolysaccharide from E. coli (Sigma-Aldrich), chicken egg white lysozyme (Sigma-Aldrich), or 0.25 μg insulin (Fitzgerald) and incubated overnight at 4 °C. The next morning, plates were washed three times using wash buffer (PBS pH 7.5, 0.001% Tween), were blocked using blocking buffer (PBS pH 7.5, 0.1% Tween-20, 1 mM EDTA, 2% BSA) for two hours at room temperature, and then were washed three times with wash buffer. Following blocking, nanobodies (200 μL) were incubated at the indicated concentrations at room temperature in PBS pH 7.5 for two hours. After three more washes, plates were incubated with HRP-anti V5 antibody (Abcam ab1325, 1:10,000 dilution) in PBS + 2% BSA for one hour at room temperature. Plates then were washed three times with wash buffer and 1-Step ABTS substrate solution (100 μL, Thermo Scientific) was added to the plates, which were then incubated in the dark for 20 min. Stop solution (1% SDS in PBS, 100 μL) was added to each plate and absorbance at 405 nm was measured using a Spectromax M5 microplate reader. Results were analyzed in GraphPad Prism.

Serum anti-dsDNA Ab levels were determined by ELISA as described previously (Kawagoe et al, 2009 (link)). Briefly, plates were coated with 5 μg/ml calf thymus dsDNA (Sigma-Aldrich). Sera were added to the plate and further incubated with AP-conjugated anti-mouse IgG Ab after washing. Then the AP substrate (Sigma-Aldrich) was added, and absorbance was measured at 405 nm. Anti-dsDNA concentrations were quantified according to the standard curve. Concentrations of total IgM and IgG1 levels in the sera were determined by ELISA as described previously (Kawagoe et al, 2009 (link)).