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We evaluated whether the cerebrospinal fluid (CSF) concentration of cartilage oligomeric matrix protein (COMP) is related to disease severity, prognosis and matrix metalloproteinase (MMP)-9 activity of the CSF in miniature dachshund with intervertebral disc herniation. Samples were obtained from 23 patients and 6 normal dogs, and all patients received hemilaminectomy. Twenty dogs recovered successfully and 3 of 11 dogs without deep nociception had MMP-9 activity in the CSF and an unsuccessful outcome. The COMP levels from patients were significantly higher than those from normal dogs. MMP-9 activity and neurological severity were not related to the COMP levels. However, the COMP levels from 3 unsuccessful cases that had MMP-9 activity were significantly lower than those from all recovered cases and/or successful cases without deep nociception. Concerning severe cases, increased proteolytic activity might affect the COMP concentration and prognosis due to MMP-9 associated deleterious effects., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Study Design: Cerebrospinal fluid (CSF) from dogs with intervertebral disc herniation (IVDH) were analyzed using a specific antibody against cartilage oligomeric matrix protein (COMP). Immunolocalization and genetic expression of COMP were also examined in the spinal cords of mouse, rat, and dog., Objective: To investigate the hypothesis that COMP is present in CSF of dogs with IVDH, and the clinical relevance of COMP expression in CSF as a potential biomarker of spinal cord injury., Summary of Background Data: In dog IVDH, diagnostic imaging is useful for determining the spinal cord compression, but less useful for assessment of traumatic degeneration. Aggrecan, a well-known component of cartilage matrix, is increased in CSF from cases of neural damage and inflammation of the spinal cord., Methods: CSF from the cisterna magna (C-CSF) and the lumbar spine (L-CSF) of 19 dogs with IVDH and 5 normal dogs were analyzed using inhibition enzyme-linked immunosorbent assay (ELISA) and immunoblotting with an antibody cross-reactive with dog COMP. Samples of normal spinal cord from mouse, rat, and dog were also prepared for immunohistochemistry and in situ hybridization., Results: ELISA values were significantly higher for L-CSF than for C-CSF in dogs with IVDH, whereas there was no significant difference between them in normal dogs. Immunoblots of L-CSF samples revealed positive bands of approximately 500 kDa in 6 cases of IVDH (positive cases), but no signal in negative cases. ELISA values were significantly higher in the positive cases than in the negative cases. Both COMP protein and mRNA were present at high levels in the gray matter of the spinal cord in all species., Conclusion: In dog IVDH, release of COMP from the spinal cord in association with injury may lead to COMP accumulation in L-CSF posterior to the site of disc extrusion, and therefore might be a predictive marker of spinal cord injury.

To investigate the expression of cartilage oligomeric matrix protein (COMP) associated with oncogenesis, samples of serum and tumor tissue were obtained from 25 dogs with tumors. The serum levels of COMP in dogs with tumors were significantly higher than in 38 normal controls and correlated with the concentrations of tumor tissue extracts. Positive bands to COMP antibody were detected on immunoblots of tumor extracts. Immunohistochemistry and in situ hybridization demonstrated positive signals of COMP and its mRNA in the cytoplasm of tumor cells from mammary gland tumors, but not in the normal mammary gland. Positive immunohistochemistry results were also obtained for COMP in mast cell tumor and melanoma cells. Oncogenesis might augment production of COMP and the serum level of COMP in dogs.

Reasons for Performing Study: Measurement of cartilage oligomeric matrix protein (COMP) in serum has potential for diagnosis of equine osteoarthritis (OA), but clinical use is currently limited by the lack of specificity of an inhibition ELISA as well as by baseline increases due to exercise. Improved methods for ELISA with increased antigen specificity and sensitivity are therefore required for reliable measurement., Hypothesis: Measurement of the serum level of COMP by sandwich ELISA allows identification of horses with OA., Methods: New monoclonal antibodies (mAbs) were elicited against equine cartilage COMP, their epitopes were determined and a sandwich ELISA was developed. The concentrations of COMP in synovial fluid (SF; n=100) and sera (n=100) from OA cases were measured by sandwich ELISA as well as by inhibition ELISA and compared with concentrations in normal joints (n=95) and horses (n=50)., Results: Immunoblots of enzymatically cleaved COMP showed that the new mAbs recognised different epitopes located on a 20 kDa fragment between K63 and K238 of the EGF-like repeats. Inhibition ELISA with any mAb detected significantly increased levels of COMP in OA SF compared with normal SF, whereas no significant difference was detected between serum levels of COMP in OA and normal horses. Conversely, sandwich ELISA with the combination of unlabelled 2A11 x biotinylated 11F10 mAbs detected a significant increase in COMP levels in both serum and SF from OA cases compared with levels in normal animals., Conclusions and Potential Relevance: Measurement of serum COMP with sandwich ELISA may be useful in identifying horses with OA.

We investigated the relationship between cartilage oligomeric matrix protein (COMP) levels in synovial fluid (SF), serum and urine and the development of osteochondral damage and osteophyte (OP) formation following intraarticular fractures of the carpus in racehorses in order to assess the clinical usefulness of COMP as a diagnostic biomarker of developmental osteoarthritis (OA). Two monoclonal antibodies (mAb clones 2A11 and 3C8) raised against equine COMP were shown to be capable of detecting the molecule in serum and urine as well as SF. Fifty-one samples were obtained from 26 OP-positive (OP(+)) and 25 OP-negative (OP(-)) racehorses with carpal bone fracture, in whom OP was ascertained arthroscopically and radiographically. The COMP measurements obtained using the two mAbs were highly correlated with each other in SF, serum, or urine. Horses with OP(+) showed a significantly higher [urinary COMP (microg)]/[urinary creatinine (mg)] ratio (4.94 +/- 5.10 and 1.46 +/- 1.19, using mAbs 2A11 and 3C8, respectively) than OP(-) horses (2.80 +/- 1.72 and 0.93 +/- 0.49, respectively). The relationship between serum and urine COMP levels and the period from injury to surgery were extrapolated using a polynomial expression. Measurement of COMP, especially in urine, has potential as a predictive marker of advanced OA following carpal bone fractures in racehorses.

Objective: To investigate the effects of polysulfated glycosaminoglycan (PSGAG) treatment on serum cartilage oligomeric matrix protein (COMP) concentration, matrix metal-loproteinase-2 (MMP-2) and -9 (MMP-9) activities, C-reactive protein (CRP) concentration, and lameness scores in dogs with osteoarthritis., Animals: 16 dogs with osteoarthritis and 5 clinically normal dogs., Procedures: Dogs with osteoarthritis had a history of chronic lameness, and osteophytes were observed on radiographic evaluation of the affected joint. Polysulfated glycosaminoglycan was administered IM twice a week for a total of 8 treatments to all dogs with osteoarthritis and to clinically normal control dogs., Results: Lameness scores after PSGAG treatment in osteoarthritic dogs improved in 12 of the 16 dogs. Serum COMP concentrations in osteoarthritic dogs were significantly higher than in control dogs before treatment. Lameness scores in osteoarthritic dogs decreased significantly after treatment, compared with before treatment. Lameness scores of 9 dogs with hind limb lameness improved significantly after treatment; these dogs had corresponding decreases in serum COMP concentrations. After treatment, serum COMP concentrations and lameness scores of 7 dogs with forelimb lameness remained high and were significantly higher than those of dogs with hind limb lameness. Serum MMP-9 activities of dogs with forelimb lameness were significantly higher than in dogs with hind limb lameness after treatment., Conclusions and Clinical Relevance: IM administration of PSGAG inhibited COMP degradation in dogs with osteoarthritis. Results indicate that decreases in serum COMP concentrations might be related to improvement in lameness after PSGAG treatment.

Objective: To evaluate changes in serum cartilage oligomeric matrix protein (COMP) concentrations in response to exercise in horses., Animals: 15 horses in experiment 1 and 27 horses in experiment 2., Procedures: In experiment 1, 15 Thoroughbreds free of orthopedic disease underwent a standardized exercise protocol. Running velocity and heart rate (HR) were recorded, and blood samples were collected immediately before (baseline) and 1, 5, and 24 hours after a single episode of exercise. In experiment 2, 27 horses underwent 9 stages of a training program in which each stage consisted of 4 to 8 consecutive daily workouts followed by a rest day. Blood samples were collected immediately before the first and final daily workouts in each stage. Serum COMP concentrations were measured via inhibition ELISA with a monoclonal antibody (14G4) against equine COMP., Results: In experiment 1, mean serum COMP concentration was significantly higher than baseline 1 and 5 hours after exercise and returned to baseline concentrations 24 hours after exercise. Mean serum baseline COMP concentration increased as the velocity of running at maximum HR and at an HR of 200 beats/min increased, being significantly higher during the third and fourth exercise tests than during the first. In experiment 2, mean baseline COMP concentration at the final workout of each stage was significantly higher than that at the first workout, beginning with stage 3., Conclusions and Clinical Relevance: Serum COMP concentrations changed significantly in response to exercise. Exercise may enhance movement of COMP into the circulation as well as change the basal turnover rate of COMP.

Objective: To quantify the urinary concentration of cartilage oligomeric matrix protein (COMP), and to evaluate the relationship between urinary COMP concentration and the catabolic activity of synovial fluid (SF) in diseased horses., Methods: COMP in horse urine was detected by immunoblotting with a monoclonal antibody (mAb; 14G4) raised against equine COMP from articular cartilage. Urine and serum samples were obtained from 83 Thoroughbred horses with aseptic joint diseases (AJD, 79 horses) or septic joint diseases (SJD, four horses) at the time of anesthesia induction, and samples of SF were obtained during surgery. Control samples of urine (n=111) were collected from normal horses free of any orthopedic diseases after they had been racing. COMP concentration was determined in all samples using inhibition enzyme-linked immunosorbent assay (ELISA) with mAb 14G4. SF samples were also used for the quantification of gelatinase activity., Results: Positive bands of COMP fragments were determined on the immunoblots with mAb 14G4. The urinary COMP concentrations in AJD and SJD horses (1.02+/-0.75 and 1.55+/-1.17 microg/100mg creatinine, respectively) were significantly higher than normal (0.57+/-0.29 microg/100mg creatinine). In 55 horses with fractures in the AJD group there was a logarithmic relationship (r=-0.45, P<0.001) between the urinary and SF COMP measurements, while the urinary COMP level was positively correlated with matrix metalloproteinase (MMP)-2 and -9 activities (r=0.30, P<0.05 and r=0.51, P<0.001, respectively) in SF., Conclusions: The urinary COMP assay with mAb 14G4 is useful for discriminating horses with osteoarthritis. The higher COMP levels in urine from such horses would be indicative of enhanced proteolytic activity, in addition to the increased COMP levels in the diseased joints.

Reasons for Performing Study: Cartilage oligomeric matrix protein (COMP) is abundant within cartilage; its turnover and/or degradation have been investigated in various equine joint diseases and it has been suggested that COMP fragmentation might be useful for monitoring such conditions., Objectives: To determine whether COMP metabolism is compromised in equine osteoarthritis (OA) and whether COMP degradation is a useful joint marker representing cartilage destruction., Hypothesis: A monoclonal antibody (mAb) with a higher affinity for degraded COMP allows discrimination of diseased joints by quantifying COMP levels and fragmentation., Methods: A mAb (clone14G4) was generated against equine cartilage COMP. The NH2-terminal sequence of enzyme-cut COMP fragments recognised by 14G4 was determined, as was the efficiency of binding to COMP (using a generated COMP peptide). COMP concentration and fragmentation were analysed in synovial fluid (SF) from normal horses and those with OA., Results: The mAb 14G4 had a higher affinity for the smaller fragments of equine COMP, compared with a mAb (clone 12C4) generated against human COMP. The 14G4 epitope was identified as between C134 and F147. The COMP values in OA (mean +/- s.d. 205.8 +/- 90.9 microg/ml) were significantly higher than in the normal (133.1 +/- 31.5 microg/ml) SF. On the immunoblots of OA sample, the proportions of intact COMP were significantly lower, while smaller fragments ranging from 75 to 290 kDa were higher compared with the normal SF., Conclusions and Potential Relevance: The mAb 14G4 reliably detects COMP degradation as well as synthesis, and fragmentation analysis combined with quantification in SF could be useful to study equine OA.

This study was designed to assay and compare cartilage oligomeric matrix protein (COMP) in horse sera, in samples from normal and joint diseased horses, and to investigate the relationships between COMP in sera and synovial fluids (SF) with keratan sulphate (KS) data. Sera from 38 horses free of any joint pathology (controls) and from horses with aseptic joint disease (AJD horses, n = 40) were assayed for COMP and KS concentrations. Of the 78 horses in the study, 53 were also assayed for COMP and KS concentrations in SF. COMP and KS were measured by inhibition ELISA, using monoclonal antibodies 12C4 and 5D4, respectively. The COMP concentration in sera from AJD horses (mean +/- s.d. 10.7 +/- 7.4 microg/ml) was significantly (P<0.02) lower than in control sera (14.8 +/- 7.8 microg/ml). The joint disease sera also had significantly lower (P<0.01) KS levels (180.5 +/- 61.8 ng/ml) than controls (237.1 +/- 116.1 ng/ml). A significant correlation (r = 0.52, n = 53, P<0.001) was seen between serum and SF in COMP levels; no such relationship was seen in KS levels. It is possible that serum COMP concentration could be a more specific marker of equine joint disease than any other described to date.

Objective: To assay concentrations of cartilage oligomeric matrix protein (COMP) in canine sera and synovial fluid (SF), to compare COMP concentrations in clinically normal dogs and dogs with joint disease, and to analyze changes in COMP concentrations in dogs with experimentally induced acute synovitis., Animals: 69 control dogs without joint disease, 23 dogs with naturally occurring aseptic arthropathy, and 6 dogs with experimentally induced synovitis., Procedure: Serum (n = 69) and SF (36) were obtained from control dogs. Samples of serum (n = 23) and SF (13) were obtained from dogs with naturally occurring aseptic arthropathy with or without radiographic features of osteoarthritis (OA). Serum and SF were obtained before and 1, 2, 3, and 7 days after induction of synovitis. The COMP concentrations were determined by use of an inhibition ELISA that had canine cartilage COMP and monoclonal antibody against human COMP., Results: Concentrations of COMP in serum and SF of control dogs were 31.3+/-15.3 and 298.7+/-124.7 microg/ml, respectively. In naturally occurring OA, COMP concentrations in serum (44.9+/-177 microg/ml) and SF (401.7+/-74.3 microg/ml) were significantly higher than corresponding concentrations in control dogs. The COMP concentration in SF peaked 24 and 48 hours after induction of synovitis, whereas concentration in serum peaked on day 3., Conclusions and Clinical Relevance: These results supported the hypothesis that COMP concentration in serum and SF of dogs may be altered after cartilage degradation or synovitis. Measurement of COMP concentrations can be useful when differentiating arthropathies in dogs.

Objective: This study was designed to assay cartilage oligomeric matrix protein (COMP) in equine synovial fluids and to compare the concentration in synovial fluids from normal horses with joint diseased horses. The relationship between the COMP degradation and the matrix metalloproteinase activity in synovial fluids was also investigated., Design: Using COMP antigen prepared from equine articular cartilage and murine monoclonal antibody (12C4) raised against human COMP, an inhibition ELISA was developed. COMP in equine synovial fluids from normal and diseased joints was quantified. Metalloproteinase activities were evaluated in the same synovial fluids by a gelatin degradation ELISA. COMP fragments were evaluated qualitatively by Western blotting., Results: The COMP inhibition ELISA was reliable at concentrations of equine COMP between 62.5 and 2000 ng/ml. COMP values in joint fluids in both aseptic and septic joint disease (19.7+/-15.3 and 16.1+/-11.2 microg/ml, respectively) were significantly (P < 0.001) lower than normal (53.2+/-29.0 microg/ml). The molecular sizes of COMP on immunoblots were different between normal and diseased synovial fluids; more fragments were seen in diseased fluids. The aseptic (26.6 +/- 20.6%) and septic joint disease synovial fluids (36.1 +/- 37.5%) had significantly higher (P < 0.02 and 0.002, respectively) gelatinolytic activities than normal (13.6 +/- 13.7%). There was a negative correlation (R = -0.31, P < 0.002) between COMP level and gelatinase activity. Conclusions We conclude that the fragment pattern and the absolute COMP concentration maybe useful for monitoring joint disease, and that COMP degradation in synovial fluids from progressed joint disease may be due to MMP gelatinolytic activity., (Copyright 2001)