Use of two-dimensional gel electrophoresis to measure changes in synovial fluid proteins from patients with rheumatoid arthritis treated with antibody to CD4

Synovial fluid proteins from microliter volumes of synovial fluid were reso lved by two-dimensional polyacrylamide gel electrophoresis and detected by silver staining to investigate the feasibility of using two-dimensional (2D ) electrophoresis in the clinical research setting and provide global disea se information of disease progression. Several hundred proteins could be re solved as spots, many of which displayed the characteristic pattern of plas ma-derived glycoproteins, The lowest level of detection was approximately 0 .2 ng from a total of 50 mug of protein loaded. Most of the proteins could be identified on the basis of pi and molecular weight when compared with pl asma protein maps on the World Wide Web. Unknown proteins were characterize d by mass spectrometry of tryptic digests and by comparison with peptide da tabases. Synovial fluids from patients with rheumatoid arthritis were analy zed using this technique, Each subject received a fixed dose of antibody to CD4 as part of a phase II clinical trial to determine the efficacy of this immunosuppressive treatment in modifying disease activity. Synovial fluid was removed at day 0, followed by administration of antibody. Subsequent re moval of synovial fluid and additional administration of antibody were carr ied out at different times thereafter, Changes in levels of acute-phase pro teins were quantified by densitometry of silver-stained 2D polyacrylamide g els. Other parameters of disease progression such as serum C-reactive prote in and physician's global assessment of clinical condition were used for co mparison. In this way, changes in acute-phase proteins towards normal level s, as measured by 2D polyacrylamide gel electrophoresis, could be correlate d with clinical improvement and conventional clinical chemistry measurement s. Thus, the system can be used for quantitative analysis of protein expres sion in sites of autoimmune disease activity such as the synovial fluid of rheumatoid arthritis patients.