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Quantitation of repetitive epitopes in glycosaminoglycans immobilized on hydrophobic membranes treated with cationic detergents

På svenska

Author

  • M Rosen
  • I Edfors-Lilja
  • Sven Björnsson

Summary, in English

Glycosaminoglycans (GAGs) are linear carbohydrate polymers containing repetitive sequences of differently sulfated uronic acid and glycosamine residues that are recognized by antibodies raised against proteoglycans. We have developed a method to demonstrate such repetitive sequence motifs in isolated GAG chains immobilized on hydrophobic membranes derivatized with cationic detergents. Six monoclonal antibodies directed against Cs (2B6, 3B3, Cs56, and 1B5), Hs (HepSS), and Ks (5134) were used to detect native and chondroitinase-generated epitopes in the immobilized GAGs. All antibodies, except 1B5, were able to detect epitopes in both proteoglycans and isolated GAGs. Type of detergent and buffer composition affected the accessibility and the retention of immobilized GAGs. The epitope density, i.e., the number of repetitive epitopes per GAG mass, was estimated as the ratio between antibody (epitope) and Alcian blue (mass) staining measured simultaneously. The epitope profiles, using six antibodies, were different for each sample (CsA, CsC, Ds, Hs, intact cartilage, and human serum). The epitope profile may be used as a structural characteristic of a GAG population. Electrophoretic separation of GAGs based on their glucuronic/ioduronic acid content and O-sulfate/N-sulfate ratio was performed using a diethylene glycol-diaminobutanol agarose gel. The electrophoretic populations were characterized by immunoblotting to detergent-treated membranes.

Publishing year

2002

Language

English

Pages

210-222

Publication/Series

Analytical Biochemistry

Volume

308

Issue

2

Document type

Journal article

Publisher

Elsevier

Topic

  • Medicinal Chemistry

Keywords

  • immobilization
  • membrane bound
  • solid phase
  • cationic detergent
  • glycosaminoglycan
  • proteoglycan
  • chondroitin sulfate
  • dermatan sulfate
  • heparan sulfate
  • epitopes
  • sequences
  • alcian blue
  • dot blot
  • transfer
  • blotting

Status

Published

Research group

  • Clinical Chemistry, Malmö

ISBN/ISSN/Other

  • ISSN: 1096-0309