At the end of the animal experiments, serum were collected and levels of specific antibodies were measured by enzyme-linked immunosorbent assay (ELISA).
Serum titers of anti-CII or anti-SRBC IgG were determined as previously described51 (link)54 (link). Briefly, 96-well ELISA plate were coated with bovine CII (50 μg/ml) or extracted SRBC membranes (20 μg/ml) (prepared as described previously55 (link)) overnight at 4 °C. Each diluted serum sample was added and incubated. Horse-radish peroxidase (HRP) conjugated goat anti-mouse IgG (H + L), IgG1and IgG2a Abs (Invitrogen, San Diego, CA, USA) were used. The optical density is measured spectrophotometrically at 450 nm.
Cytokines in sera and culture supernatants were detected using mouse IL-6, IL-17A (BD Biosciences, San Diego, CA) and IL-21 (R&D systems, Minneapolis, MN) ELISA kits according to the manufacturer’s instructions.
Serum titers of anti-CII or anti-SRBC IgG were determined as previously described51 (link)54 (link). Briefly, 96-well ELISA plate were coated with bovine CII (50 μg/ml) or extracted SRBC membranes (20 μg/ml) (prepared as described previously55 (link)) overnight at 4 °C. Each diluted serum sample was added and incubated. Horse-radish peroxidase (HRP) conjugated goat anti-mouse IgG (H + L), IgG1and IgG2a Abs (Invitrogen, San Diego, CA, USA) were used. The optical density is measured spectrophotometrically at 450 nm.
Cytokines in sera and culture supernatants were detected using mouse IL-6, IL-17A (BD Biosciences, San Diego, CA) and IL-21 (R&D systems, Minneapolis, MN) ELISA kits according to the manufacturer’s instructions.
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