OBJECTIVE: To develop an enzyme linked immunosorbent assay (ELISA) to quantify the levels of specific aggrecan fragments generated by aggrecanase-mediated cleavage at the 373Glu-374 Ala bond within the aggrecan interglobular domain. METHODS: The ELISA employs a commercially available monoclonal antibody to capture aggrecan fragments containing keratan sulfate (KS). Aggrecan fragments generated by cleavage at the Glu-Ala bond were then detected using a monoclonal neoepitope antibody (mAb OA-1) that specifically recognizes the N-terminal sequence 'ARGSVIL'. RESULTS: The mAb OA-1 antibody was highly specific for the immunizing neoepitope peptide since neither peptides spanning the cleavage site nor mutated peptides were detected. Aggrecan fragments generated by ADAMTS-4 digested human aggrecan monomers and from IL-1-stimulated human cartilage explants were quantified by the ELISA, and we observed increased sensitivity of the ELISA compared to mAb OA-1 Western analysis. We also observed that the basal, as well as IL-1-stimulated production of ARGS aggrecan fragments from human articular cartilage explants was blocked by a selective aggrecanase inhibitor, consistent with generation of the ARGS neoepitope in human articular cartilage being mediated by aggrecanase. Using purified human aggrecan digested by ADAMTS-4 as standard to quantify ARGS aggrecan fragments in human synovial fluids, we determined that the calculated amount of ARGSVIL-aggrecan fragments by ELISA measurement is in agreement with the published levels of these fragments, supporting its potential utility as a biomarker assay for osteoarthritis. CONCLUSION: We have developed an assay that detects and quantifies specific aggrecan fragments generated by aggrecanase-mediated cleavage. Because aggrecanase mediates degradation of human articular aggrecan in joint disease, the KS/mAb OA-1 ELISA may serve as a biomarker assay for evaluation of preclinical and clinical samples.
OBJECTIVE: To develop an enzyme linked immunosorbent assay (ELISA) to quantify the levels of specific aggrecan fragments generated by aggrecanase-mediated cleavage at the 373Glu-374 Ala bond within the aggrecan interglobular domain. METHODS: The ELISA employs a commercially available monoclonal antibody to capture aggrecan fragments containing keratan sulfate (KS). Aggrecan fragments generated by cleavage at the Glu-Ala bond were then detected using a monoclonal neoepitope antibody (mAb OA-1) that specifically recognizes the N-terminal sequence 'ARGSVIL'. RESULTS: The mAb OA-1 antibody was highly specific for the immunizing neoepitope peptide since neither peptides spanning the cleavage site nor mutated peptides were detected. Aggrecan fragments generated by ADAMTS-4 digested human aggrecan monomers and from IL-1-stimulated human cartilage explants were quantified by the ELISA, and we observed increased sensitivity of the ELISA compared to mAb OA-1 Western analysis. We also observed that the basal, as well as IL-1-stimulated production of ARGS aggrecan fragments from humanarticular cartilage explants was blocked by a selective aggrecanase inhibitor, consistent with generation of the ARGS neoepitope in humanarticular cartilage being mediated by aggrecanase. Using purified human aggrecan digested by ADAMTS-4 as standard to quantify ARGS aggrecan fragments in human synovial fluids, we determined that the calculated amount of ARGSVIL-aggrecan fragments by ELISA measurement is in agreement with the published levels of these fragments, supporting its potential utility as a biomarker assay for osteoarthritis. CONCLUSION: We have developed an assay that detects and quantifies specific aggrecan fragments generated by aggrecanase-mediated cleavage. Because aggrecanase mediates degradation of human articular aggrecan in joint disease, the KS/mAb OA-1 ELISA may serve as a biomarker assay for evaluation of preclinical and clinical samples.
Authors: J Larkin; T A Lohr; L Elefante; J Shearin; R Matico; J-L Su; Y Xue; F Liu; C Genell; R E Miller; P B Tran; A-M Malfait; C C Maier; C J Matheny Journal: Osteoarthritis Cartilage Date: 2015-03-20 Impact factor: 6.576
Authors: Morten A Karsdal; Thasia Woodworth; Kim Henriksen; Walter P Maksymowych; Harry Genant; Philippe Vergnaud; Claus Christiansen; Tanja Schubert; Per Qvist; Georg Schett; Adam Platt; Anne-Christine Bay-Jensen Journal: Arthritis Res Ther Date: 2011-04-28 Impact factor: 5.156
Authors: Ellen van der Aar; Henri Deckx; Sonia Dupont; Ann Fieuw; Stephane Delage; Staffan Larsson; André Struglics; L Stefan Lohmander; Agnes Lalande; Emilie Leroux; David Amantini; Paul Passier Journal: Clin Pharmacol Drug Dev Date: 2021-12-02
Authors: Reuben Gobezie; Alvin Kho; Bryan Krastins; David A Sarracino; Thomas S Thornhill; Michael Chase; Peter J Millett; David M Lee Journal: Arthritis Res Ther Date: 2007 Impact factor: 5.156
Authors: Rebecca Mae Black; Yang Wang; André Struglics; Pilar Lorenzo; Viveka Tillgren; Martin Rydén; Alan J Grodzinsky; Patrik Önnerfjord Journal: Osteoarthr Cartil Open Date: 2020-09-05