HMGB1 serum levels were quantitatively measured using an ELISA kit (IBL International GmbH, Hamburg, Germany) according to the manufacturer’s instructions about test procedures. Collected samples from JIA subgroups, ReA group, and healthy controls were centrifuged within 4 h at 2500×g for 10 min at 4 °C and frozen/stored at − 80 °C until used to perform the ELISA. Plates coated with anti-HMGB1 polyclonal antibody were incubated with serum samples for 22 h at 37 °C. Next, peroxidase-conjugated anti-HMGB1, 2-monoclonal antibody was applied as detection. The analytical sensitivity was 0.2 ng/mL in the highly sensitive range. Cross-reactivity with HMGB2 was less than 2% and below the lower limit of detection.
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