Background Aggrecan degradation is believed to be a crucial process in joint diseases such as OA and RA. The ADAMTS cleavage site TEGE373-374ARGS is a hot spot for investigation of aggrecan degradation. We have developed a competitive ELISA AGNx1 for detecting fragments containing neo-epitope NITEGE373, and a sandwich ELISA G1-373 for detecting fragment containing both NITEGE373 and an epitope located in G1/G2 domain of aggrecan. There are disadvantages for the existing ELISAs: lower sensitivity for the fragment in serum samples and unsatisfied technical performance result for G1-373 with higher coefficient of variation (CV) variation.

Objectives The aim of this study was to develop a sandwich ELISA 356-373 with higher sensitivity detecting fragments containing NITEGE373.

Methods The ELISAs were characterized and compared by using the Full Depth Cartilage (FDC) explant culture model. FDC from superficial zone to the deep zone of the cartilage explants was obtained from the joint of one to two-year- old calf. During 21 days culture, serum free medium containing oncostatin M and tumor necrosis factor alpha were replaced very second or third day and store at -20°C before analysis by: (1) competitive NITEGE373 ELISA (AGNx1) detecting aggrecan fragments containing neo-epitope NITEGE373, (2) sandwich G1-373 ELISA detecting aggrecan fragments containing both neo-epitope NITEGE373 and G1 domain, (3) sandwich 356-373 ELISA detecting fragments containing both neo-epitope NITEGE373 and epitope 352TVQTVTW358 in interglobular domain (IGD).

Results (1) Aggrecanase-derived aggrecan fragments containing NITEGE373 were dose-dependently released in the early (day 2-7 of culturing, 43% of total release) and mid phase (day 9-14, 54% of total release) into the supernatant from FDC explants stimulated with catabolic cytokines. (2) The fragments released, profiled by the three assays, were similar but different: the NITEGE373 competitive ELISA and the 356-373 sandwich ELISA showed two peak releases. The G1-373 sandwich ELISA only showed one release. (3) The release of NITEGE373 fragments detected by NITEGE373 competitive ELISA or the 356-373 sandwich ELISA was delayed by MMPs inhibitor GM6001, whereas the fragments release detected with G1-373 sandwich ELISA was partially but significantly inhibited by GM6001 (33% inhibition), indicating that MMPs are involved in G1-373 ELISA-detecting fragment derivation. (4) Higher sensitivity and better technical performance data was achieved for the 356-373 ELISA compared to NITEGE373 competitive ELISA and G1-373 ELISA, respectively. (5) 356-373 ELISA could be applied for human synovial fluid testing with good sensitivity (sample diluted 1:8), specificity (signal inhibited to 14% by peptide with only one epitope of the sandwich assay) and dilution recovery (mean Rec 99% within 1:4 and 1:64 dilution), indicating its further potential clinical application for joint diseases as a biomarker.

Conclusions We developed a new sandwich ELISA detecting the ADAMTSs derived fragments containing neo-epitope NITEGE373 and another epitope 352TVQTVTW358 in IGD. This ELISA is more sensitive than the previous competitive NITEGE373 ELISA, which lack sensitivity for human samples test, and more technically robust than the G1-373 ELISA which has weak stability.

Disclosure of Interest None Declared