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

Struglics, André LU ; Larsson, Staffan LU ; Björklund, Maria LU and Lohmander, Stefan LU (2009) In Osteoarthritis and Cartilage 17. p.497-506
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Osteoarthritis and Cartilage
volume
17
pages
497 - 506
publisher
Elsevier
external identifiers
  • wos:000264975000012
  • pmid:19095471
  • scopus:61549089342
ISSN
1063-4584
DOI
10.1016/j.joca.2008.09.017
language
English
LU publication?
yes
id
2b8faf68-d35a-4e44-be10-f89ef90cd603 (old id 1276072)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/19095471?dopt=Abstract
date added to LUP
2009-01-09 12:58:52
date last changed
2017-07-23 04:50:34
@article{2b8faf68-d35a-4e44-be10-f89ef90cd603,
  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.},
  author       = {Struglics, André and Larsson, Staffan and Björklund, Maria and Lohmander, Stefan},
  issn         = {1063-4584},
  language     = {eng},
  pages        = {497--506},
  publisher    = {Elsevier},
  series       = {Osteoarthritis and Cartilage},
  title        = {Western blot quantification of aggrecan fragments in human synovial fluid indicates differences in fragment patterns between joint diseases.},
  url          = {http://dx.doi.org/10.1016/j.joca.2008.09.017},
  volume       = {17},
  year         = {2009},
}