Sandwich ELISA system for cartilage oligomeric matrix protein in equine synovial fluid and serum.
BACKGROUND
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.
Yamanokuchi
K
K
Department of Veterinary Clinical Science, Kagoshima University, 21-24 Korimoto 1-chome, Kagoshima 890-0065, Japan.
Tagami
M
M
Nishimatsu
E
E
Shimizu
Y
Y
Hirose
Y
Y
Komatsu
K
K
Misumi
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015415:Biomarkers / Q000032:analysis; D016022:Case-Control Studies; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D018604:Epitope Mapping / Q000662:veterinary; D016326:Extracellular Matrix Proteins / Q000032:analysis / Q000097:blood / Q000276:immunology; D005260:Female; D006023:Glycoproteins / Q000032:analysis / Q000097:blood / Q000276:immunology; D006734:Horse Diseases / Q000097:blood / Q000175:diagnosis; D006736:Horses; D064235:Matrilin Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D010003:Osteoarthritis / Q000097:blood / Q000175:diagnosis / Q000662:veterinary; D012680:Sensitivity and Specificity; D013582:Synovial Fluid / Q000737:chemistry / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D015415:Biomarkers; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; C577540:Matn1 protein, mouse; D064235:Matrilin Proteins