Background The depletion of aggrecan in articular cartilage is an early event in the pathogenesis of Osteoarthritis and Rheumatoid arthritis. Both ADAMTS-4 and ADAMTS-5 are believed to play important roles in the generation of high-molecular-weight fragments of aggrecan. Several assays are available for measuring these aggrecanase-derived degradation fragments (e.g. NITEGE and ARGSV) in synovial fluid and blood. These are indirect measures of the aggrecanase activity. There are presently no immuno-assays available for the direct measurement of aggrecanase activity. The ADAMTS-4 propeptide is cleaved from N-terminal upon activation of the enzyme resulting in a NeoEpitope on the ADAMTS-4, which is specific for the active form of ADAMTS-4. We hypothesize that the NeoEpitope of active ADAMTS-4, measured in synovial fluid and blood, will be a sensitive biomarker of joint deterioration in OA and RA
Objectives To develop and test a novel immuno-assay for detection of the specific NeoEpitope of active ADAMTS-4 in synovial fluid and blood
Methods The peptide FASLSR was conjugated to KLH and immunized for Balb/C mice. The best mouse with reaction against NeoEpitope was selected to generate hybridoma by standard method. The best MAb was chosen based on specificity towards NeoEpitope but not elongated and nonsense peptide. Furthermore, the MAb was in vitro characterized by western blot using active ADAMTS-4 from bovine cartilage explants supernatants and active ADAMTS-4 from a commercial source. A competition ELISA was developed using this MAb. Bovine cartilage explants were cultured in the presence of TNF and OSM for 21 days and changed the media every 2 or 3 days. At day 7, 14 and 21, the supernatants were collected and the extracellular matrix proteins were extracted. The active ADAMTS-4 released into supernatants and retained in the matrix from cartilage explants were analyzed by our ELISA and assay for aggrecan neoepitope NITEGE373. The assay was applied to detect the release of active ADAMTS-4 from human serum (n=8) and also from the synovial fluid of patients (n=10)
Results After fusion and serial subcloning, one MAb was obtained and characterized. It had specific response to the specific peptide, but not toward the elongated peptide. Furthermore this MAb was specific for active ADAMTS4 based on the molecular weight of single band on western blot. The aggrecan specific neoepitope NITEGE373was induced early in the presence of stimulators and peak at day14 then return to the background level. The active ADAMTS-4 retained in the matrix approach the highest level at day 14 simultaneously. And the active ADAMTS-4 in supernatant increased at late stage of stimulation by catabolic factors. We hypothesize that active ADAMTS-4 was bound tightly to the cartilage matrix at the early stage and then was released from the matrix when aggrecan was depleted from the explants. The levels of active ADAMTS-4 were detected in human blood within the range of 0.2-0.5ng/ml and 0.03-0.5ng/ml for synovial fluid.
Conclusions We identified and developed a new competitive ELISA for measuring active ADAMTS-4, which can detect active ADAMTS-4 at protein level and therefore provide us more information of post-translational regulation of ADAMTS-4. Moreover this NeoEpitope can be measured in human blood and synovial fluid and will be a potential biomarker for joint degenerative diseases.
Disclosure of Interest None Declared