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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/403909
- author
- Pratta, M. A.
; Su, J. L.
; Leesnitzer, M. A.
; Struglics, André
LU
; Larsson, Staffan
LU
; Lohmander, Stefan
LU
and Kumar, S.
- organization
- publishing date
- 2006
- 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
- 2016-04-01 11:40:22
- date last changed
- 2024-01-07 16:03:31
@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}},
keywords = {{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}},
doi = {{10.1016/j.joca.2006.01.012}},
volume = {{14}},
year = {{2006}},
}