Calprotectin

Blood samples were collected at convalescent time points between 0.9 and 11.3 years after KD onset when all patients were generally healthy. Blood was collected and separated immediately by centrifugation and stored at −80°C. We measured EDTA plasma levels of calprotectin, Gal‐3, sST2, GDF‐15, and serum levels of PIPC by ELISA according to the manufacturer's instructions: calprotectin, Gal‐3, GDF‐15: R&D Systems, Minneapolis, MN, USA, sST2: Critical Diagnostics, San Diego, CA, USA and PIPC: Quidel, San Diego, CA, USA.

Blood samples from KD subjects were collected at convalescent time points (median: 14.5 years; range: 0.9–55.0 years post-KD onset). Blood was collected and separated immediately by centrifugation and stored at −80 °C. We measured EDTA plasma levels of calprotectin, Gal-3, sST2, GDF-15, and serum levels of PIPC by ELISA according to the manufacturer’s instructions: calprotectin, Gal-3, GDF-15: R & D Systems, Minneapolis, MN, USA, sST2: Critical Diagnostics, San Diego, CA, USA and PIPC: Quidel, San Diego, CA, USA.

Plasma levels of calprotectin (R&D Systems, Minneapolis, MN, USA) and immune complexes (ICs) (MicroVue CIC-C1q EIA, Quidel, Athens, OH, USA) were measured by ELISA, following the manufacturers’ instructions. The IC ELISA is based on the capacity of complement factor C1q, immobilized to the plate, to bind to circulating ICs. Quantification of circulating NETs was performed by utilizing myeloperoxidase (MPO)–DNA ELISA as described by us [10 (link)]. First, 96-well microtitre plates (Corning Inc., Corning, NY, USA) were coated with anti-MPO antibody (4 μg/ml; Bio-Rad Laboratories, Hercules, CA, USA) overnight at 4°C, and then blocked with 1% BSA in PBS for 2 h at room temperature (RT). Then, plasma samples diluted 1:100 (MPO–DNA ELISA) were added in 1% BSA in PBS with 2 mM EDTA, and incubated overnight at 4°C. Anti-DNA–HRP from the Cell Death Detection ELISA kit (clone MCA-33; Roche, Indianapolis, IN, USA) was added as detection antibody for 2 h at RT. The reaction was developed with 3,3′,5,5′-tetramethylbenzidine (BD Biosciences, San Jose, CA, USA) for 20 min and stopped by the addition of 2 M sulphuric acid. Known concentrations of MPO–DNA complexes (rhMPO, R&D Systems, Minneapolis, MN, USA; calf thymus DNA, Trevigen, Gaithersburg, MD, USA) were utilized to construct a standard curve. Absorbance was measured by a plate reader at 450 nm (Synergy 2, BioTek, Winooski, VT, USA).