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

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 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.

• Authors: M A Pratta, J L Su, M A Leesnitzer, A Struglics, S Larsson, L S Lohmander, S Kumar
• Journal: Osteoarthritis and cartilage (Osteoarthritis Cartilage) Vol. 14 Issue 7 Pg. 702-13 (Jul 2006) ISSN: 1063-4584 [Print] England
• PMID: 16549371 (Publication Type: Journal Article)

Chemical References

• Aggrecans
• Interleukin-1
• Keratan Sulfate
• Endopeptidases
• ADAM Proteins
• Procollagen N-Endopeptidase
• ADAMTS4 Protein
• ADAMTS4 protein, human
• aggrecanase

Topics

• ADAM Proteins (metabolism)
• ADAMTS4 Protein
• Aggrecans (metabolism)
• Cartilage, Articular (enzymology)
• Endopeptidases
• Enzyme-Linked Immunosorbent Assay (methods)
• Humans
• Interleukin-1 (metabolism)
• Keratan Sulfate (metabolism)
• Procollagen N-Endopeptidase (metabolism)
• Sensitivity and Specificity
• Synovial Fluid (enzymology)