*4.6. ELISA Measurements*

The specific cytokine concentrations (IL-1β, IL-4, IL-6, IL-17, IL-23, IFN-γ and TNF-α) were measured in the blood sera and the supernatants of in vitro-cultured spleen cells using sandwich ELISA (R&D Systems, Minneapolis, MN, USA), according to the manufacturer's instructions.

The serum-concentration of rhG1 antigen-specific antibodies was measured using indirect ELISA as described earlier [27]. Briefly, 96-well ELISA plates were coated overnight at room temperature with the rhG1 antigen (0.1 μg/well in 100 μL carbonate coating buffer). After overnight incubation, plates were incubated for 1 h with 1.5% nonfat dry milk (NFDM) in PBS blocking buffer, followed by washing 5 times with 0.5% Tween in PBS. Next, diluted sera were added to the plate and incubated for 2 h at room temperature, then washed 5 times with 0.5% Tween in PBS. After that, anti-IgG1-peroxidase (BD Bioscience, San Jose, CA, USA) secondary antibody was added to the plate and incubated for 2 h at room temperature. The results were detected using orthophenylenediamine chromophore and H2O2 substrate.

The anti-CCP IgG1 and IgG2a antibody levels of sera were determined using the commercially available Immunoscan CCP Plus ELISA kit (SVAR, Malmö, Sweden) with slight modification. For the development of the reactions, we used anti-mouse-IgG1-peroxidase or anti-mouse-IgG2a-peroxidase (both from BD Bioscience, San Jose, CA, USA) secondary antibody instead of the kit's secondary reagent.
