Sandwich ELISA system for cartilage oligomeric matrix protein in equine synovial fluid and serum.
Abstract: 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. Objective: 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: Measurement of serum COMP with sandwich ELISA may be useful in identifying horses with OA.
Publication Date: 2009-03-24 PubMed ID: 19301580DOI: 10.2746/042516408x330356Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This study investigates the use of a sandwich enzyme-linked immunosorbent assay (ELISA) in measuring the levels of cartilage oligomeric matrix protein (COMP) in horse serum and synovial fluid, potentially improving the detection and diagnosis of equine osteoarthritis.
Research Goal
- The main goal of the research was to improve the methods for measuring the levels of COMP in horse serum and synovial fluid. This is important as increased levels of COMP are associated with equine osteoarthritis (OA), a common disease in horses that affects their joint mobility and function. A more sensitive and specific assay would allow for more reliable diagnosis of this disease.
Methods
- The researchers developed new monoclonal antibodies (mAbs) against equine cartilage COMP and determined their epitopes, the specific parts of an antigen that an antibody binds to.
- They then developed a sandwich ELISA, a type of ELISA that uses two antibodies to detect a specific antigen.
- The researchers assessed the effectiveness of this new method by measuring the concentrations of COMP in synovial fluid and serum from 100 cases of OA and compared these to the concentrations in normal joints and healthy horses.
Results
- The new mAbs were found to recognise different epitopes located on a 20 kDa fragment of the EGF-like repeats of COMP.
- The researchers discovered that increased levels of COMP in OA synovial fluid compared to normal fluid could be detected using any mAb via common inhibition ELISA; however, no significant difference was observed when comparing serum levels using the same method.
- Conversely, sandwich ELISA, when using a combination of the newly developed mAbs, was found to be capable of detecting elevated levels of COMP in both OA synovial fluid and OA serum, compared with the levels in normal samples.
Conclusion
- The researchers concluded that the sandwich ELISA they developed provides a more sensitive and specific method for measuring COMP levels in both serum and synovial fluid, potentially improving the diagnosis of equine OA.
Cite This Article
APA
Yamanokuchi K, Tagami M, Nishimatsu E, Shimizu Y, Hirose Y, Komatsu K, Misumi K.
(2009).
Sandwich ELISA system for cartilage oligomeric matrix protein in equine synovial fluid and serum.
Equine Vet J, 41(1), 41-46.
https://doi.org/10.2746/042516408x330356 Publication
Researcher Affiliations
- Department of Veterinary Clinical Science, Kagoshima University, 21-24 Korimoto 1-chome, Kagoshima 890-0065, Japan.
MeSH Terms
- Animals
- Antibodies, Monoclonal / immunology
- Biomarkers / analysis
- Case-Control Studies
- Enzyme-Linked Immunosorbent Assay / methods
- Enzyme-Linked Immunosorbent Assay / veterinary
- Epitope Mapping / veterinary
- Extracellular Matrix Proteins / analysis
- Extracellular Matrix Proteins / blood
- Extracellular Matrix Proteins / immunology
- Female
- Glycoproteins / analysis
- Glycoproteins / blood
- Glycoproteins / immunology
- Horse Diseases / blood
- Horse Diseases / diagnosis
- Horses
- Matrilin Proteins
- Mice
- Mice, Inbred BALB C
- Osteoarthritis / blood
- Osteoarthritis / diagnosis
- Osteoarthritis / veterinary
- Sensitivity and Specificity
- Synovial Fluid / chemistry
- Synovial Fluid / metabolism
Citations
This article has been cited 5 times.- Smith R, Önnerfjord P, Holmgren K, di Grado S, Dudhia J. Development of a Cartilage Oligomeric Matrix Protein Neo-Epitope Assay for the Detection of Intra-Thecal Tendon Disease. Int J Mol Sci 2020 Mar 20;21(6).
- Ekman S, Lindahl A, Rüetschi U, Jansson A, Björkman K, Abrahamsson-Aurell K, Björnsdóttir S, Löfgren M, Hultén LM, Skiöldebrand E. Effect of circadian rhythm, age, training and acute lameness on serum concentrations of cartilage oligomeric matrix protein (COMP) neo-epitope in horses. Equine Vet J 2019 Sep;51(5):674-680.
- Wang C, Lakshmipriya T, Gopinath SCB. Amine-Aldehyde Chemical Conjugation on a Potassium Hydroxide-Treated Polystyrene ELISA Surface for Nanosensing an HIV-p24 Antigen. Nanoscale Res Lett 2019 Jan 14;14(1):21.
- Bertuglia A, Pagliara E, Grego E, Ricci A, Brkljaca-Bottegaro N. Pro-inflammatory cytokines and structural biomarkers are effective to categorize osteoarthritis phenotype and progression in Standardbred racehorses over five years of racing career. BMC Vet Res 2016 Nov 8;12(1):246.
- Anderson JR, Phelan MM, Caamaño-Gutiérrez E, Clegg PD, Rubio-Martinez LM, Peffers MJ. Metabolomic and proteomic stratification of equine osteoarthritis. Equine Vet J 2025 Sep;57(5):1204-1218.
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