Western blot quantification of aggrecan fragments in human synovial fluid indicates differences in fragment patterns between joint diseases

Abstract

Objective: To develop a Western blot method for quantification of multiple aggrecan fragments in human synovial fluids (SFs).

Method: SF aggrecan fragments were prepared from knee healthy (reference), knee injury and arthritis subjects by CsCl gradient centrifugations collecting D1 fractions. Samples were analyzed by Western blot, using antibodies against the N-terminal epitope ARGS and the G3 domain, and fragments were quantified using a digital luminescence image analyzer.

Results: The method had a coefficients of variation of 10-30%, and a high correlation (r(S)=0.86) with a corresponding enzyme-linked immunosorbent assay (ELISA). The SFs from reference, knee injured and arthritic subjects contained two major ARGS fragments, ARGS-SELE and ARGS-CS1, and three major G3 fragments (GRGT-G3, GLGS-G3 and AGEG-G3). Compared to the reference, the acute arthritis and acute joint injury groups had a 30-fold elevated concentration of ARGS fragments, and both groups had a higher proportion of the aggrecan in joint fluid as ARGS fragments compared to the other groups. The reference and chronic injury groups had an excess of ARGS-CS1 fragments over ARGS-SELE fragments, while subjects with acute arthritis or osteoarthritis had a more even distribution between these fragments.

Conclusions: We have developed a novel Western blot quantification method for quantification of SF aggrecan fragments which can differentiate fragments of different sizes sharing the same epitope. The anti-ARGS and anti-G3 quantitative Western blots provided information important for a better understanding of the proteolytic pathways in aggrecan breakdown, information that discriminates between different joint diseases, and may aid in identification of new biomarkers.