Microtiter plates maxisorp

Serum samples were collected on days 7, 14, and 21 post immunization, and the titers of anti-CII IgG Abs were measured by ELISA. Bovine CII (1 μg/mL, Chondrex, Inc., Redmond, WA, USA) was coated onto microtiter plates (Maxisorp; Nunc, Roskilde, Denmark) overnight at 4 °C. After blocking with 1% BSA in PBS, serially diluted serum samples were added and incubated for 1 h at room temperature. After washing, HRP-conjugated rabbit anti-mouse IgG1, IgG2a, or IgG2b Ab (Zymed Laboratories, San Francisco, CA) was added and incubated for 2 h at 37 °C. After washing, Ab binding was visualized using o-phenylenediamine (Sigma-Aldrich). A standard serum composed of a mixture of sera from arthritic mice was added to each plate in serial dilutions, and a standard curve was constructed. The standard serum was defined as 1 U, and the Ab titers of serum samples were determined by the standard curve.

Bovine serum albumin (BSA), NADPH, 1-chloro-2,4-dinitrobenzene (CNDB), glutathione, o-phenylenediamine dihydrochloride (OPD), ethoxyresorufin, methoxyresorufin, benzyloxyresorufin and pentoxyresorufin, were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Microtiter plates (MaxiSorp) were purchased from Nunc (Roskilde, Denmark). Polyclonal CYP1A1 antibody was purchased from Biosense Laboratories, (Bergen, Norway). CYP2B6, CYP2E1 and CYP2B1/2 antibodies were purchased from Santa Cruz Biotechnology (Dallas TX, USA). The CYP3A antiserum was a kind gift from Dr. Malin Celander. All other chemicals were of the highest commercially available grade.