Establishment of a monoclonal antibody (1/14/16H9) for detection of equine keratan sulfate.
OBJECTIVE
To establish a sensitive and specific monoclonal antibody (MAB) against equine keratan sulfate (KS) and to develop an enzyme immunoassay for measurement of the concentration of KS in serum and synovial fluid from horses.


METHODS
18 synovial fluid and 48 serum samples were obtained from clinically normal horses and horses with arthritis.


METHODS
BALB/c mice were immunized with chondroitinase-ABC-digested proteoglycan monomer from equine joint cartilage, and MAB were raised, using Sp2/O cells as a fusion partner. A competitive ELISA was optimized, using one of the established MAB, and KS concentration in synovial fluid and sera from horses was measured.


RESULTS
The high-titer MAB1/14/16H9, which specifically recognizes the epitope on equine KS, was identified. This antibody had no reactivity with chondroitin sulfate and core protein of proteoglycan monomers, hyaluronan, heparin, dermatan sulfate, and heparan sulfate. A competitive ELISA for determination of KS concentration was optimized, using this antibody. Precision data were obtained for the range of 10 to 160 ng/ml. The within- and between-assay coefficients of variation were 10.0 and 12.7%, respectively.


CONCLUSIONS
MAB 1/14/16H9 that specifically recognized equine KS was established and was used to develop an enzyme immunoassay for measurement of the concentration of KS in synovial fluid and sera from horses. It is expected that the assay system using MAB 1/14/16H9 will contribute to evaluation of cartilage metabolic activity in horses.
Okumura
M
M
Department of Veterinary Clinical Sciences, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
Fujinaga
T
T
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D006736:Horses / Q000378:metabolism*; D007632:Keratan Sulfate / Q000032:analysis* / Q000097:blood / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D012680:Sensitivity and Specificity; D013582:Synovial Fluid / Q000737:chemistry
D000911:Antibodies, Monoclonal; D007632:Keratan Sulfate