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Measurement of inflammatory biomarkers in synovial tissue extracts by enzyme-linked immunosorbent assay.

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 1beta (IL-1beta), 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 < or = 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-1beta, 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.

Arthritis, Rheumatoid↗

[Immunodiffusion analysis of embryonic prealbumin in a tumorous tissue extract].

By immunodiffusion analysis the presence of embryonal prealbumin (EPA) in extracts of different tumors has been studied. EPA is most frequently detected in extracts of bone tumors (75.0%) with the titre up to 1 : 256 and in tumors of soft tissues (74.5%) also with the titre up to 1 : 256. The EPA immunochemical test is believed to be of value for monitoring tumors of the connective-tissue origin.

Bone Neoplasms↗

The use of plasma desorption time-of-flight mass spectrometry to screen for products of prohormone processing in crude tissue extracts.

Californium-252 plasma desorption mass spectrometry (252Cf PDMS) of a crude, desalted, extract of piscine endocrine pancreas provided mass information for the major biologically active peptide hormones present in this tissue. An extraction procedure compatible with 252Cf PDMS analysis was developed. In extracts of catfish pancreas, strong molecular ions were identified in the positive mode for somatostatin-14 (1638 amu), O-glycosylated somatostatin-22 (2944 amu), glucagon (3512 amu), glucagon-like peptide (3785 amu), insulin (ca. 5550 amu), and other prohormone-derived peptides. Both protonated species and sodium adducts were apparent in the mass spectrum. A number of other molecular ions were observed including somatostatin-26, 1-10 (1014 amu) and the entire portion of prosomatostatin-22 remaining after removal of somatostatin-22 (6465 amu). The data obtained by this method also resulted in the identification of the third major product of proglucagon processing in catfish pancreas, glicentin-related polypeptide. Subtractive Edman degradation analyzed by 252Cf PDMS was also used to confirm a mass assignment.

Amino Acid Sequence↗