2.3. ELISA-based measurement of serum IL-27 levels

Serum IL-27 levels were detected using a human IL-27 enzyme-linked immunosorbent assay (ELISA) kit (e-Bioscience, San Diego, CA, USA), according to the manufacturer’s protocol. Generally, serum samples and standard solutions were incubated with antibody-coated plates according to the manufacturer’s protocol. Subsequently, 50 µL of diluted biotin-conjugate, 100 µL of diluted streptavidin–HRP, and 100 µL of TMB substrate solution were subsequently incubated at room temperature for 2 h, 1 h, and 30 min, respectively. Stop solution of 100 µL was used to stop the reaction. The sample was directly used for detection without dilution and was repeated three times to obtain the average value. The optical density was read at 450 nm using a microplate reader (SYNERGY 2; BioTek, USA). Serum IL-27 levels were determined according to the standard curves. The minimum threshold concentration of IL-27 was 9.5 pg/mL.