Pro-inflammatory markers: CRP, IL-6, TNF-α

Pro-inflammatory biomarkers of CRP, IL-6, and TNF-α were measured using intravenous blood samples. Venous blood was collected directly after the cognitive function test. If the blood test and the cognitive function test could not be performed on the same day, the blood test was performed the day after the cognitive function test. Analyses of biomarker concentrations were performed for all participants simultaneously.

High-sensitivity plasma levels of CRP were measured in duplicate by ELISA based on purified protein and polyclonal anti-CRP antibodies (N-Assay LA CRP-s, Nittobo, Japan). IL-6 (Human IL-6 Immunoassay HS600B, R&D Systems, USA) and TNF-α (Human TNF-α Immunoassay HSTA00E, R&D Systems, USA) levels were measured in duplicate by a high sensitivity enzyme-linked immunosorbent assay (ELISA).