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

SummaryObjectiveTo develop an enzyme linked immunosorbent assay (ELISA) to quantify the levels of specific aggrecan fragments generated by aggrecanase-mediated cleavage at the 373Glu–374Ala bond within the aggrecan interglobular domain.MethodsThe 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’.ResultsThe 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.ConclusionWe 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.