Development of a Cartilage Oligomeric Matrix Protein Neo-Epitope Assay for the Detection of Intra-Thecal Tendon Disease

The diagnosis of tendon injury relies on clinical signs and diagnostic imaging but imaging is subjective and does not always correlate with clinical signs. A molecular marker would potentially offer a sensitive and specific diagnostic tool that could also provide objective assessment of healing for the comparison of different treatments. Cartilage Oligomeric Matrix Protein (COMP) has been used as a molecular marker for osteoarthritis in humans and horses but assays for the protein in tendon sheath synovial fluids have shown overlap between horses affected by tendinopathy and controls. We hypothesized that quantifying a COMP neoepitope would be more discriminatory of injury. COMP fragments were purified from synovial fluids of horses with intra-thecal tendon injuries and media from equine tendon explants, and mass spectrometry of a consistent and abundant fragment revealed a ~100 kDa COMP fragment with a new N-terminus at the 78th amino-acid (NH2-TPRVSVRP) located just outside the junctional region of the protein. A competitive inhibition ELISA based on a polyclonal antibody raised to this sequence yielded more than a 10-fold rise in the mean neoepitope levels for tendinopathy cases compared to controls (5.3 ± 1.3 µg/mL (n = 7) versus 58.8 ± 64.3 µg/mL (n = 13); p = 0.002). However, there was some cross-reactivity of the neoepitope polyclonal antiserum with intact COMP, which could be blocked by a peptide spanning the neoepitope. The modified assay demonstrated a lower concentration but a significant > 500-fold average rise with tendon injury (2.5 ± 2.2 ng/mL (n = 6) versus 1029.8 ± 2188.8 ng/ml (n = 14); p = 0.013). This neo-epitope assay therefore offers a potentially useful marker for clinical use

Quantitative proteomics at different depths in human articular cartilage reveals unique patterns of protein distribution

The articular cartilage of synovial joints ensures friction-free mobility and attenuates mechanical impact on the joint during movement. These functions are mediated by the complex network of extracellular molecules characteristic for articular cartilage. Zonal differences in the extracellular matrix (ECM) are well recognized. However, knowledge about the precise molecular composition in the different zones remains limited. In the present study, we investigated the distribution of ECM molecules along the surface-to-bone axis, using quantitative non-targeted as well as targeted proteomics.\ In a discovery approach, iTRAQ mass spectrometry was used to identify all extractable ECM proteins in the different layers of a human lateral tibial plateau full thickness cartilage sample. A targeted MRM mass spectrometry approach was then applied to verify these findings and to extend the analysis to four medial tibial plateau samples. In the lateral tibial plateau sample, the unique distribution patterns of 70 ECM proteins were identified, revealing groups of proteins with a preferential distribution to the superficial, intermediate or deep regions of articular cartilage. The detailed analysis of selected 29 proteins confirmed these findings and revealed similar distribution patterns in the four medial tibial plateau samples. The results of this study allow, for the first time, an overview of the zonal distribution of a broad range of cartilage ECM proteins and open up further investigations of the functional roles of matrix proteins in the different zones of articular cartilage in health and disease

The skeletal phenotype of chondroadherin deficient mice

Chondroadherin, a leucine rich repeat extracellular matrix protein with functions in cell to matrix interactions, binds cells via their a2b1 integrin as well as via cell surface proteoglycans, providing for different sets of signals to the cell. Additionally, the protein acts as an anchor to the matrix by binding tightly to collagens type I and II as well as type VI. We generated mice with inactivated chondroadherin gene to provide integrated studies of the role of the protein. The null mice presented distinct phenotypes with affected cartilage as well as bone. At 3–6 weeks of age the epiphyseal growth plate was widened most pronounced in the proliferative zone. The proteome of the femoral head articular cartilage at 4 months of age showed some distinct differences, with increased deposition of cartilage intermediate layer protein 1 and fibronectin in the chondroadherin deficient mice, more pronounced in the female. Other proteins show decreased levels in the deficient mice, particularly pronounced for matrilin-1, thrombospondin-1 and notably the members of the a1-antitrypsin family of proteinase inhibitors as well as for a member of the bone morphogenetic protein growth factor family. Thus, cartilage homeostasis is distinctly altered. The bone phenotype was expressed in several ways. The number of bone sialoprotein mRNA expressing cells in the proximal tibial metaphysic was decreased and the osteoid surface was increased possibly indicating a change in mineral metabolism. Micro-CT revealed lower cortical thickness and increased structure model index, i.e. the amount of plates and rods composing the bone trabeculas. The structural changes were paralleled by loss of function, where the null mice showed lower femoral neck failure load and tibial strength during mechanical testing at 4 months of age. The skeletal phenotype points at a role for chondroadherin in both bone and cartilage homeostasis, however, without leading to altered longitudinal growth

Development of fluorescence based flow immunoassays utilising restricted access columns

The development of high-speed flow immunoassay techniques is described. The principles are based on heterogeneous flow immunoassay interactions. High sample throughput can be used for screening small analytes in a number of biological matrices originating from samples of water from environmentally polluted areas, or biological fluids such as urine and plasma. The immunochemical detection principle is based on chromatographic separation of the immunocomplex formed (AbAg or AbAg*) and the free antigen (Ag) by a restricted access (RA) column, utilising size-exclusion and reversed phase mechanisms. A fluorescein-labelled analyte (Ag*) was used in the competitive assay format with fluorescence detection. Sample throughput was 80 h-1 and detection limits 1.4 nM (300pgml-1) for atrazine and 2.3 nM (500 pg ml-1) for the sum of triazines. Analyses could be performed at a sample throughput of 400 6 h-1 shift. Basic immunoaffinity interactions of a number of immunoreagents, using fluorescence polarisation were studied and outlined both for triazines and for 2,4-D. Structural variations in tracer synthesis confirmed that this is an important part in the design and optimisation of flow immuno methodologies

Biomarkers and proteomic analysis of osteoarthritis.

Our friend and colleague, Dr. Dick Heinegård, contributed greatly to the understanding of joint tissue biochemistry, the discovery and validation of arthritis-related biomarkers and the establishment of methodology for proteomic studies in osteoarthritis (OA). To date, discovery of OA-related biomarkers has focused on cartilage, synovial fluid and serum. Methods, such as affinity depletion and hyaluronidase treatment have facilitated proteomics discovery research from these sources. Osteoarthritis usually involves multiple joints; this characteristic makes it easier to detect OA with a systemic biomarker but makes it hard to delineate abnormalities of individual affected joints. Although the abundance of cartilage proteins in urine may generally be lower than other tissue/sample sources, the protein composition of urine is much less complex and its collection is non-invasive thereby facilitating the development of patient friendly biomarkers. To date however, relatively few proteomics studies have been conducted in OA urine. Proteomics strategies have identified many proteins that may relate to pathological mechanisms of OA. Further targeted approaches to validate the role of these proteins in OA are needed. Herein we summarize recent proteomic studies related to joint tissues and the cohorts used; a clear understanding of the cohorts is important for this work as we expect that the decisive discoveries of OA-related biomarkers rely on comprehensive phenotyping of healthy non-OA and OA subjects. Besides the common phenotyping criteria that include, gender, age, and body mass index (BMI), it is essential to collect data on symptoms and signs of OA outside the index joints and to bolster this with objective imaging data whenever possible to gain the most precise appreciation of the total burden of disease. Proteomic studies on systemic biospecimens, such as serum and urine, rely on comprehensive phenotyping data to unravel the true meaning of the proteomic results

Quantification of cartilage oligomeric matrix protein (COMP) and a COMP neoepitope in synovial fluid of patients with different joint disorders by novel automated assays

Objective: To develop automated immunoassays for the quantification of Cartilage Oligomeric Matrix Protein (COMP) and a COMP neoepitope in synovial fluid and to investigate their diagnostic potential in different joint conditions. Methods: Two sandwich immunoassays were developed for the quantification of COMP and a COMP neoepitope, using an automated analyser (IDS-iSYS, Immunodiagnostic Systems, Boldon, UK). Assay performance was evaluated in terms of sensitivity, recovery, linearity, and intra- and inter-assay precision. Clinical performance was evaluated by analysing synovial fluid from patients diagnosed with rheumatoid arthritis (RA), reactive arthritis (ReA), osteoarthritis (OA) or acute trauma (AT). Results: Both automated assays showed good performance for all parameters tested. Quantification of these biomarkers showed the highest median values for Total COMP in the OA group, followed by the AT group, the ReA group, and the RA group. For the COMP neoepitope the AT group showed the highest median value, followed by the ReA group, the OA group, and the RA group. The ratio COMP neoepitope/Total COMP showed distinct differences between the patients groups, as well as between RA patients with slow or rapid progression of joint damage. Conclusions: The newly developed automated assays have a good technical performance, can reliably quantify different epitopes on the COMP molecule and show different levels of the two biomarkers in synovial fluid in patients with different joint diseases. The combination of these two assays, measuring their ratio, shows promise for early detection of patients with RA with different prognosis regarding progression of joint damage

Cartilage matrix remodelling differs by disease state and joint type

Dramatic alterations in mechanical properties have been documented for osteoarthritic (OA) cartilage. However, the matrix composition underlying these changes has not been mapped and their aetiology is not entirely understood. We hypothesised that an understanding of the cartilage matrix heterogeneity could provide insights into the origin of these OA-related alterations. We generated serial transverse cryo sections for 7 different cartilage conditions: 2 joint sites (knee and hip), 2 disease states (healthy and OA) and 3 tissue depths (superficial, middle and deep). By laser capture microscopy, we acquired ~200 cartilage matrix specimens from territorial (T) and interterritorial (IT) regions for all 7 conditions. A standardised matrix area was collected for each condition for a total of 0.02 ± 0.001 mm3 (corresponding to 20 µg of tissue) from a total of 4800 specimens. Extracted proteins were analysed for abundance by targeted proteomics. For most proteins, a lower IT/T ratio was observed for the OA disease state and knee joint type. A major cause of the altered IT/T ratios was the decreased protein abundance in IT regions. The collagenase-derived type III collagen neo-epitope, indicative of collagen proteolysis, was significantly more abundant in OA cartilage. In addition, it was enriched on average of 1.45-fold in IT relative to T matrix. These results were consistent with an elevated proteolysis in IT regions of OA cartilage, due to degenerative influences originating from synovial tissue and/or produced locally by chondrocytes. In addition, they offered direct evidence for dynamic remodelling of cartilage and provided a cogent biochemical template for understanding the alterations of matrix mechanical properties