A novel method to quantify TSG‐6 protein levels in synovial fluid from osteoarthritis patients

Abstract

TSG‐6, the protein product of Tumor Necrosis Factor‐stimulated gene‐6, is a potential biomarker of disease activity/progression in osteoarthritis (OA). By ELISA, TSG‐6 is elevated in synovial fluid (SF) from OA patients and its catalytic activity, for the covalent transfer of inter‐α‐inhibitor heavy chains onto hyaluronan (HA), is reported to correlate with progression to total knee replacement. Here, quantitative western blot (qWB) analysis, following sequential digestion with hyaluronidase and chondroitinase enzymes, was developed for the accurate determination of TSG‐6 levels in SFs. qWB analysis of 125 OA SFs yielded values of 1.0–45.2 μg/mL (median: 3.9 μg/mL), 100–1000‐fold higher than those previously reported; TSG‐6 concentration was positively correlated with age and pain/outcome scores in male patients. The values determined, by ELISA or a heavy chain transfer activity assay, following the same digestion conditions (in 23 SFs), were both ≥10‐fold lower. Apparent TSG‐6 catalytic activity was significantly affected by the concentration of HA present in SF or added exogenously. Thus, the interactions of TSG‐6 with components of OA SF prevent accurate determination of its concentration/activity when using existing assays. However, the quantitative western blot method developed here appears more suitable for further evaluation of the utility of TSG‐6 as a biomarker in OA.