To validate a modified ligand-binding assay for the detection of aggrecanase generated aggrecan fragments with the ARGS neoepitope in synovial fluid (SF) and blood, and to verify the identity of aggrecan fragments found in blood.
An enzyme-linked immunosorbent assay (ELISA) on the Meso Scale Discovery (MSD) platform for detection of ARGS-aggrecan was validated, using a standard made from recombinant human aggrecan. Matched samples of SF, serum, plasma, and urine were obtained from 36 subjects at different time points after knee injury, and analysed for ARGS-aggrecan content. Aggrecan was purified from serum and plasma pools and analysed by Western blot.
The limits of quantification for the ARGS-aggrecan assay was between 0.2 and 0.025 pmol ARGS/ml, and the sensitivity of the assay was improved two-fold compared to when using a standard purified from human donors. The ARGS concentrations were highest in SF (mean, range; 3.02, 0.36-30.22 pmol/ml), 20 times lower in the blood samples (0.14, 0.055-0.28 pmol/ml serum and 0.13, 0.053-0.28 pmol/ml plasma), and 80 times lower in urine (0.036, below detection - 0.087 pmol/ml). Serum-ARGS and plasma-ARGS concentrations were similar, and correlated (r(S) = 0.773, P < 0.001). SF concentration correlated with serum concentrations (r(S) = 0.420, P = 0.011). In blood, we identified 129-138 kDa aggrecan fragments containing the ARGS neoepitope.
This novel ARGS-aggrecan assay is highly sensitive and suited for analysis of SF and blood samples. Both SF and blood contains ARGS-aggrecan, and ARGS concentrations in SF and serum are correlated.