ABSTRACT
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.
Subject(s)
Collagen Type VI/blood , Enzyme-Linked Immunosorbent Assay/methods , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Spondylitis, Ankylosing/pathology , Adolescent , Adult , Aged , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Biomarkers/blood , Biomarkers/metabolism , Case-Control Studies , Collagen Type VI/metabolism , Female , Humans , Male , Mice , Mice, Inbred BALB C , Middle Aged , Peptide Fragments , Prognosis , Proteolysis , Retrospective Studies , Spondylitis, Ankylosing/diagnosis , Spondylitis, Ankylosing/metabolism , Young AdultABSTRACT
AIM: To measure levels of the collagen V formation marker CO5-1230 during liver fibrosis progression and regression. METHODS: Monoclonal antibodies were raised against the sequence TAALGDIMGH located at the start of the C-terminal propeptide between amino acid position 1230' and 1239' (CO5-1230). An assay developed using the biotin-streptavidin system was evaluated in a rat reversible model of fibrosis. Animals were treated for duration of 4, 6 and 8 weeks. Animals that were treated for 8 weeks were left to regress for a period of 14, 20 and 26 weeks. RESULTS: Mean CO5-1230 level for control animals was found to be 8.7 ng/mL. CO5-1230 marker levels, at termination points, for CCl(4) treated animals was be 8.7 ng/mL at 4 weeks (P < 0.05, ROC: 0.83), 11.4 ng/mL at 6 weeks (P < 0.001, ROC: 0.93) and 10.8 ng/mL at 8 weeks (P < 0.05, ROC: 0.82). During regression phase, marker levels were statistically significantly decreased when compared with the marker levels at 8 weeks of treatment. Marker levels were found to be 5.9 ng/mL (P < 0.001, ROC: 0.8) after 14 weeks of regression, 3.9 ng/mL (P < 0.001, ROC: 0.95) after 20 weeks and 4.5 ng/mL (P < 0.001, ROC: 0.97) after 26 weeks of regression. CONCLUSIONS: The data indicates that CO5-1230 levels are statistically significantly increased when CCl(4) intoxication stimulus is applied in all treatment time points. CO5-1230 levels return back to control levels when the stimulus is removed. The above finding adds to our previous evaluation of the marker and suggests that CO5-1230 may be a promising potential marker for liver fibrosis staging and monitoring in both disease progression and regression.
ABSTRACT
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.
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Epitopes/immunology , Peptide Fragments/blood , Peptide Fragments/immunology , Procollagen/blood , Procollagen/immunology , Aged , Amino Acid Sequence , Animals , Blotting, Western , Bone Density Conservation Agents/pharmacology , Calibration , Clone Cells , Demography , Diphosphonates/administration & dosage , Diphosphonates/pharmacology , Female , Humans , Ibandronic Acid , Molecular Sequence Data , Osteocalcin/blood , Ovariectomy , Peptide Fragments/chemistry , Placebos , Postmenopause/blood , Postmenopause/drug effects , Procollagen/chemistry , RatsABSTRACT
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.
