Maxisorp 96 well immunoplates

To assess hIL-10R-1 binding, a competitive displacement ELISA was used. Maxisorp 96-well immunoplates (Nunc, Roskilde, Denmark) were incubated overnight with 400 ng·mL⁻¹ of hIL-10 in coating buffer (15 mM Na²CO₃, 35 mM NaHCO₃, pH 9.6) at 4 °C. Plates were washed then blocked with 10% bovine serum albumin in PBS for 1 h. Samples of IL-10 serially diluted in PBS containing 0.4% BSA and 0.02% Tween20 were incubated with 300 ng·mL⁻¹ hIL-10R1–Fc chimeric protein (R&D Systems, Minneapolis, MN, USA) in non-absorbent plates at 25 °C for 1 h. The mixture was transferred to plates coated with hIL-10 and incubated at 25 °C for 1 h to capture the unbound hIL-10R1–Fc protein. The captured hIL-10R1–Fc protein was detected with a goat anti-human IgG-HRP conjugate (Thermo Fisher Scientific, Waltham, MA, USA) and 3,3′,5,5′-tetramethylbenzidine substrate (BD Biosciences). The reaction was stopped with H₂SO₄ and quantified by measuring the absorbance at 450 nm. The percentage change in hIL-10R1-Fc protein bound to the immobilised hIL-10 was calculated relative to that detected in the absence of soluble IL-10.

A modified enzyme-linked immunosorbant assay (ELISA) was used to assess binding of the recombinant IL-10s for IL-10R1. Maxisorp 96-well immunoplates (Nunc, Roskilde, Denmark) were incubated with 400 ng/mL of hIL-10 in coating buffer (15 mm Na²CO₃, 35 mm NaHCO₃, pH 9.6) at 4°C for 16 h and blocked with 1% bovine serum albumin (BSA) and 0.02% Tween 20 at 37°C for 45 min. Plates were washed between steps with wash buffer. Samples of hIL-10 or eIL-10, serially diluted in binding buffer (NaCl / Pi with 0.4% BSA and 0.02% Tween 20), were incubated with 300 ng/mL human IL-10R1–Fc chimeric protein (R&D Systems, Minneapolis, MN, USA) in non-absorbent plates at 25°C for 1 h. The mixture was then transferred to plates coated with hIL-10 and incubated at 25°C for 1 h to capture the unbound hIL-10R1–Fc protein. The captured IL-10R1–Fc protein was detected by biotinylated anti-human Ig (Dako, Glostrup, Denmark) and streptavidin-peroxidase (Sigma) and tetramethylbenzidine substrate reagent and quantified by measuring the absorbance at 450 nm. The percentage change in IL-10R1-Fc protein bound was then calculated relative to that detected in the absence of soluble IL-10.