Abstract
Western blot of synovial fluid has been widely used for osteoarthritis (OA) research and diagnosis, but there is no ideal loading control for this purpose. Although β-actin is extensively used as loading control in western blot, it is not suitable for synovial fluid because it is not required in synovial fluid as a cytoskeletal protein. A good loading control for synovial fluid in OA studies should have unchanged content in synovial fluids from normal and OA groups, because synovial fluid protein content can vary with changes in synovial vascular permeability with OA onset. In this study, we explore the potential of using α1-antitripsin (A1AT) as loading control for OA synovial fluid in western blot. A1AT level is elevated in inflammatory conditions such as rheumatoid arthritis (RA). Unlike RA, OA is a non-inflammation disease, which does not induce A1AT. In this study, we identified A1AT as an abundant component of synovial fluid by Mass Spectrometry and confirmed that the level of A1AT is relative constant between human OA and normal synovial fluid by western blot and ELISA. Hence, we proposed that A1AT may be a good loading control for western blot in human OA synovial fluid studies provided that pathological conditions such as RA or A1AT deficiency associated liver or lung diseases are excluded.
Highlights
Synovial fluid has been widely used for research, diagnosis, and treatment of joint diseases, such as osteoarthritis (OA)
Previous study has demonstrated that A1AT, a serine protease inhibitor synthesized in the liver, is highly abundant in both blood serum and synovial fluid
Using the quantitative radial immunodiffusion method, Swedlund et al found that A1AT contents in normal and OA synovial fluid are very close [9,12]
Summary
Synovial fluid has been widely used for research, diagnosis, and treatment of joint diseases, such as osteoarthritis (OA). We are the first lab to explore the potential of using α1-antitripsin (A1AT) as loading control for synovial fluid in western blot. A1AT balances the activity of neutrophil elastase [3], which is released by neutrophils to digest damaged cells and bacteria in response to inflammation and infection [4]. A1AT blocks apoptosis in lung endothelial cells by inhibiting caspase-3 activity [3]. A1AT deficiency can lead to lung damage by overactivated neutrophil elastase and caspase-3 [5]. In rheumatoid arthritis (RA), A1AT level in synovial fluid is significantly elevated compared to normal synovial fluid [9], which is consistent with the fact that RA involves chronic, systemic inflammation, and the presence of neutrophils in RA synovial fluid [10]
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