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Development and characterization of a highly specific and sensitive sandwich ELISA for detection of aggrecanase-generated aggrecan fragments

Pratta, M. A.; Su, J. L.; Leesnitzer, M. A.; Struglics, André LU ; Larsson, Staffan LU ; Lohmander, Stefan LU and Kumar, S. (2006) In Osteoarthritis and Cartilage 14(7). p.702-713
Abstract
Objective: To develop an enzyme linked immunosorbent assay (ELISA) to quantify the levels of specific aggrecan fragments generated by aggrecanase-mediated cleavage at the (373)Glu-(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... (More)
Objective: To develop an enzyme linked immunosorbent assay (ELISA) to quantify the levels of specific aggrecan fragments generated by aggrecanase-mediated cleavage at the (373)Glu-(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. (C) 2006 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
osteoarthritis, cartilage, synovial fluid, aggrecanase, aggrecan, biomarker
in
Osteoarthritis and Cartilage
volume
14
issue
7
pages
702 - 713
publisher
Elsevier
external identifiers
  • wos:000238895400011
  • scopus:33744908602
ISSN
1063-4584
DOI
10.1016/j.joca.2006.01.012
language
English
LU publication?
yes
id
f3556e2f-9718-4690-92a9-bb41a961e90a (old id 403909)
date added to LUP
2007-10-14 18:02:17
date last changed
2019-08-07 01:38:32
@article{f3556e2f-9718-4690-92a9-bb41a961e90a,
  abstract     = {Objective: To develop an enzyme linked immunosorbent assay (ELISA) to quantify the levels of specific aggrecan fragments generated by aggrecanase-mediated cleavage at the (373)Glu-(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. (C) 2006 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.},
  author       = {Pratta, M. A. and Su, J. L. and Leesnitzer, M. A. and Struglics, André and Larsson, Staffan and Lohmander, Stefan and Kumar, S.},
  issn         = {1063-4584},
  keyword      = {osteoarthritis,cartilage,synovial fluid,aggrecanase,aggrecan,biomarker},
  language     = {eng},
  number       = {7},
  pages        = {702--713},
  publisher    = {Elsevier},
  series       = {Osteoarthritis and Cartilage},
  title        = {Development and characterization of a highly specific and sensitive sandwich ELISA for detection of aggrecanase-generated aggrecan fragments},
  url          = {http://dx.doi.org/10.1016/j.joca.2006.01.012},
  volume       = {14},
  year         = {2006},
}