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.

• Authors: A Struglics, S Larsson, M Hansson, L S Lohmander
• Journal: Osteoarthritis and cartilage (Osteoarthritis Cartilage) Vol. 17 Issue 4 Pg. 497-506 (Apr 2009) ISSN: 1522-9653 [Electronic] England
• PMID: 19095471 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Aggrecans
• Endopeptidases
• aggrecanase

Topics

• Adult
• Aged
• Aggrecans (metabolism)
• Blotting, Western (methods)
• Endopeptidases (metabolism)
• Enzyme-Linked Immunosorbent Assay
• Female
• Humans
• Knee Injuries (metabolism)
• Knee Joint (metabolism)
• Male
• Middle Aged
• Osteoarthritis, Knee (metabolism)
• Synovial Fluid (metabolism)
• Young Adult