Au400 biochemical analyzer

Fasting blood samples were collected from all individuals and were immediately centrifuged. The plasma samples were stored at −80 °C until analysis. Serum levels of CRP were determined by an immuno-turbidimetric technique using an Olympus AU 400 biochemical analyzer (Olympus Optical, Tokyo, Japan). HLA-B27 was detected by flow cytometry using an IOTest HLA-B27-FITC/HLA-B7-PE (Beckman Coulter—Immunotech SAS, Marseille, France) and a BDTM HLA-B27 Kit (BD Bioscience, San Jose, CA, USA).

All samples were stored at − 80 °C until use. Serum levels of C-reactive protein (CRP) were determined by an immuno-turbidimetric technique using an Olympus AU 400 biochemical analyzer (Olympus Optical, Tokyo, Japan), and erythrocyte sedimentation rate (ESR) was measured according to the Fahreus and Westergren method. ANAs were detected using indirect immunofluorescence on HEP2 cells, and the autoantibodies of the ENA complex (anti-U1RNP, anti-Ro, anti-La, anti-DNA-topoisomerase I, anti-Jo-1, anti-P protein, anti-Sm, and anti-centromere) were assayed by immunoblot. Plasma levels of Hsp90 were assessed by a high-sensitivity ELISA kit (eBioscience, Vienna, Austria) according to the manufacturer's protocol. The assay recognizes human Hsp90 alpha. The calculated sensitivity is 0.03 ng/mL. The absorbance value was established at 450 nm by an ELISA reader (SUNRISE; Tecan, Grödig, Austria).