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Clinical and Diagnostic Laboratory Immunology, November 2003, p. 1002-1010, Vol. 10, No. 6
1071-412X/03/$08.00+0     DOI: 10.1128/CDLI.10.6.1002-1010.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Measurement of Inflammatory Biomarkers in Synovial Tissue Extracts by Enzyme-Linked Immunosorbent Assay

Sanna Rosengren, Gary S. Firestein, and David L. Boyle*

Center for Innovative Therapy, Division of Rheumatology, Allergy, and Immunology, University of California—San Diego School of Medicine, La Jolla, California

Received 20 June 2003/ Returned for modification 23 July 2003/ Accepted 19 August 2003

We developed methods for measuring inflammatory biomarkers (cytokines, chemokines, and metalloproteinases) in synovial biopsy specimens from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Soluble extracts of synovial fragments were prepared with mild detergent and analyzed by enzyme-linked immunosorbent assay (ELISA) for interleukin 1ß (IL-1ß), IL-6, IL-8, tumor necrosis factor alpha (TNF-{alpha}), and matrix metalloproteinase 3. The optimal detergent was 0.1% Igepal CA-630, which interfered minimally with ELISA detection but extracted 80% of IL-6 from synovial tissue. Upon spiking, 81 to 107% of added biomarkers could be recovered. To determine within-tissue variability, multiple biopsy specimens from each RA synovial extract were analyzed individually. A resulting coefficient of variation of 35 to 62% indicated that six biopsy specimens per synovial extract would result in a sampling error of <=25%. Preliminary power analysis suggested that 8 to 15 patients per group would suffice to observe a threefold difference before and after treatment in a serial biopsy clinical study. The previously described significant differences in IL-1ß, IL-6, IL-8, and TNF-{alpha} levels between RA and OA could be detected, thereby validating the use of synovial extracts for biomarker analysis in arthritis. These methods allow monitoring of biomarker protein levels in synovial tissue and could potentially be applied to early-phase clinical trials to provide a preliminary estimate of drug efficacy.

* Corresponding author. Mailing address: UCSD School of Medicine, Division of Rheumatology, Allergy, and Immunology, Mail Code 0656, 9500 Gilman Dr., La Jolla, CA 92093. Phone: (858) 822-0784. Fax: (858) 534-2606. E-mail: dboyle{at}ucsd.edu.

Clinical and Diagnostic Laboratory Immunology, November 2003, p. 1002-1010, Vol. 10, No. 6
1071-412X/03/$08.00+0     DOI: 10.1128/CDLI.10.6.1002-1010.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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