Bm blue pod

The target proteins human IL-17A (R&D Systems), human IL-17B (Peprotech), human IL-17C (R&D Systems), human IL-17D (Peprotech), human IL-17E (Peprotech), human IL-17F (AbD Serotec), mouse IL-17A (R&D Systems), rat IL-17A (Akron Biotech), canine IL-17A (R&D Systems), human IL-6 (R&D Systems), human TNF-α (Piercenet), ovalbumin (Sigma), bovine serum albumin (BSA, Sigma) cynomolgus IL-17A (produced in-house), extra domain-B of fibronectin (25 (link)) were coated onto MaxiSorp® plates (Nunc) at a concentration of 10 μg/ml. After blocking of residual binding sites with 2% milk in PBS (v/w, Rapilait, Migros, Switzerland), 2C1 monomer was incubated at 80 nm with the antibody 9E10 (Roche Applied Science, 3 μg/ml) in 2% milk in PBS. After washing, the wells were incubated with goat anti-mouse IgG in 2% milk in PBS (1:1000, Sigma, HRP-conjugated). After washing, BM Blue POD (Roche Applied Science) was added as substrate for color development. This reaction was stopped after 5 min with 1 m sulfuric acid (Sigma). Binding to human IL-17C was determined in a separate experiment, indicated by the dashed line (Fig. 2D). For each condition, the reference-subtracted absorbance was calculated by A₄₅₀ − A₆₅₀ (reference wavelength) using a SpectraMax® M5e Microplate Reader (Molecular Devices). The data were imported into Excel (Microsoft), and the average of the duplicates was calculated.

All four 2C1-Fc fusion proteins were tested in ELISA competition experiments to determine their capabilities to block the interaction between IL-17A and its receptor. Maxisorp plates (Nunc) were coated with commercially available IL-17R-Fc fusion (R&D Systems) overnight at a concentration of 0.6 μg/ml. After blocking with 1% BSA in PBS, different concentrations of the Fc fusions were pre-incubated with 2.5 nm of biotinylated IL-17A (R&D Systems; biotinylated in-house with NHS-PEG4-biotin according to the manufacturer's instructions (Pierce)); and added to the wells. After washing, the wells were incubated with streptavidin-HRP conjugate (R&D Systems) 1:200 diluted in 1% BSA/PBS. After washing, BM Blue POD (Roche Applied Science) was added as substrate for color development. This reaction was stopped after 5 min with 1 m sulfuric acid (Sigma). For each condition, the reference-subtracted absorbance was calculated by A₄₅₀ − A₆₅₀ (reference wavelength) using a SpectraMax® M5e Microplate Reader (Molecular Devices). IC₅₀ values were calculated using Prism software (version 5).

For the detection of soluble sIL-6R in sera of mice, the samples were diluted 1:15 in 1% BSA–PBS. The ELISA was performed as described67 (link). In brief, microtiter plates (Greiner Microlon) were coated with anti-murine IL-6-R pAB AF1830 (0.8 μg ml⁻¹; R&D Systems) in PBS. After blocking, 100 μl aliquots of cell lysates or culture supernatants were added. IL-6R bound to the plate was detected by biotinylated goat anti-mouse IL-6-R pAB BAF1830 (working concentration, 0.2 μg ml⁻¹; R&D Systems) followed by streptavidin–horseradish peroxidase (R&D Systems). The enzymatic reaction was performed with soluble peroxidase substrate (BM blue POD from Roche) at 37 °C and the absorbance was read at 450 nm on a SLT Rainbow plate reader (Tecan). For the detection of murine IL-6 in sera of mice, the samples were diluted 1:3 with 1% BSA–PBS.