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


CONCLUSIONS
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.
Department of Musculoskeletal Diseases, GlaxoSmithKline Pharmaceutical Company, Collegeville, PA 19426-0989, USA. micheal_a_pratta@gsk.com
M A
MA
Pratta
J L
JL
Su
M A
MA
Leesnitzer
A
A
Struglics
S
S
Larsson
L S
LS
Lohmander
S
S
Kumar
D051722:ADAM Proteins / Q000378:metabolism; D000071121:ADAMTS4 Protein; D053674:Aggrecans / Q000378:metabolism*; D002358:Cartilage, Articular / Q000201:enzymology*; D010450:Endopeptidases; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007375:Interleukin-1 / Q000378:metabolism; D007632:Keratan Sulfate / Q000378:metabolism; D011348:Procollagen N-Endopeptidase / Q000378:metabolism; D012680:Sensitivity and Specificity; D013582:Synovial Fluid / Q000201:enzymology
D053674:Aggrecans; D007375:Interleukin-1; D007632:Keratan Sulfate; D010450:Endopeptidases; D051722:ADAM Proteins; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein; C000603918:ADAMTS4 protein, human; C083641:aggrecanase