MMP mediated type V collagen degradation (C5M) is elevated in ankylosing spondylitis.

OBJECTIVES: Type V collagen has been demonstrated to control fibril formation. The aim of this study was to develop an ELISA capable of detecting a fragment of type V collagen generated by MMP-2/9 and to evaluate the assay as biomarker for ankylosing spondylitis (AS). DESIGN AND METHODS: A fragment unique to type V collagen and generated by both MMP-2/9 cleaved at the amino acid position 1317 (C5M) was selected for ELISA development. 40 AS patients and 40 age-matched controls were evaluated. RESULTS: An ELISA detecting C5M with inter- and intra-assay variations of 9.1% and 4.4% was developed. C5M levels were significantly higher in AS patients compared to controls, 229% (p<0.0001). The diagnostic AUC was 83%. CONCLUSIONS: This ELISA is the first for detecting type V collagen degradation. AS patients had highly elevated levels of MMP mediated type V collagen degradation. The prognostic and diagnostic values need to be further investigated in additional clinical settings.

Investigation of two novel biochemical markers of inflammation, matrix metalloproteinase and cathepsin generated fragments of C-reactive protein, in patients with ankylosing spondylitis.

OBJECTIVES: Ankylosing spondylitis (AS) is a chronic inflammation of the spine and the sacroiliac joints. Current markers of inflammation, such as C-reactive protein (CRP), are reflecting the production of an acute phase reactant rather than tissue specific inflammation, but the use of CRP as a diagnostic and prognostic marker for AS has not provided the sought accuracy and specificity. We hypothesized that local enzymatic activity in the disease-affected tissue, which is associated with extensive tissue turnover may, by cleavage, modify the CRP produced in the liver. These cleavage products may provide additional information on systemic inflammation as compared to that of full-length CRP. We investigated whether these CRP degradation products would provide additional diagnostic value in AS patients compared to full-length CRP. METHODS: CRP fragments were identified by mass-spectrometry. Two fragments were selected for ELISA development. One assay exclusively identified a matrix metalloproteinase (MMP) generated fragment, CRP-MMP, whereas the other assay identified a cathepsin generated fragment, CRP-CAT. Full-length CRP, CRP-MMP and CRP-CAT were measured in serum samples from 40 AS patients and 40 sex- and age-matched controls. RESULTS: Full-length CRP was not elevated in AS patients compared to controls, whereas CRP-MMP was elevated by 25% (p<0.001) and CRP-CAT by 50% (p<0.0001). The Area Under Curve of the Receiver-Operator Characteristic curve of CRP-CAT was the highest with 77%. CONCLUSIONS: MMP and cathepsin degraded CRP provided more discriminative diagnostic potential compared to that of full-length CRP in this current study. These data suggest that different pools of CRP may provide insight into the inflammation processes in AS.

Enzyme-linked immunosorbent serum assays (ELISAs) for rat and human N-terminal pro-peptide of collagen type I (PINP)--assessment of corresponding epitopes.

OBJECTIVES: The present study describes two newly developed N-terminal pro-peptides of collagen type I (PINP) competitive enzyme-linked immunosorbent assays (ELISAs) for the assessment of corresponding PINP epitopes in the rat- and human species. METHODS: Monoclonal antibodies were raised against corresponding rat and human PINP sequences and competitive assays were developed for each species. They were evaluated in relevant pre-clinical or clinical studies. RESULTS: The antibody characterizations indicated that PINP indeed was recognized. Technical robust assays were obtained. Rat PINP and tALP showed similar patterns in the gold standard osteoporosis rat ovariectomized (OVX) model. No liver contribution was observed in the liver fibrosis rat bile duct ligation model (BDL). In an osteoporosis study, the human serum PINP levels were significantly decreased after ibandronate treatment compared to placebo. CONCLUSIONS: The two corresponding PINP assays were specific and these bone turnover markers may improve translational science for the evaluation for bone-related diseases.

A novel assay for extracellular matrix remodeling associated with liver fibrosis: An enzyme-linked immunosorbent assay (ELISA) for a MMP-9 proteolytically revealed neo-epitope of type III collagen.

OBJECTIVES: Accumulation of extracellular matrix (ECM) components and increased matrix-metalloprotease (MMPs) activity are hallmarks of fibrosis. We developed an ELISA for quantification of MMP-9 derived collagen type III (CO3) degradation. DESIGN AND METHODS: A monoclonal antibody targeting a specific MMP-9 cleaved fragment of CO3 was used for development of a competitive ELISA. The assay was investigated in serum and tissues from bile duct ligated rats (BDL). RESULTS: The ELISA showed no cross-reaction with either intact CO3, or other collagens. The intra- and inter-assay CV were below 10%. Liver fibrosis was demonstrated in BDL animals by semi quantitative scoring (P<0.0001). Serum levels of CO3-610 increased 2.5 fold in BDL animals (P<0.001). The CO3-610 levels were 5 fold higher in ex vivo cultures of fibrotic livers compared to controls (P<0.001). CONCLUSION: We have developed a novel ELISA for measuring a specific fragment CO3 generated by MMP-9 important in pathogenesis of liver fibrosis.