Calf thymus histone

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.