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

Rosen, M; Edfors-Lilja, I and Björnsson, Sven LU (2002) In Analytical Biochemistry 308(2). p.210-222
Abstract
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
immobilization, membrane bound, solid phase, cationic detergent, glycosaminoglycan, proteoglycan, chondroitin sulfate, dermatan sulfate, heparan sulfate, epitopes, sequences, alcian blue, dot blot, transfer, blotting
in
Analytical Biochemistry
volume
308
issue
2
pages
210 - 222
publisher
Elsevier
external identifiers
  • wos:000178564800003
  • pmid:12419332
  • scopus:0037108658
ISSN
1096-0309
DOI
10.1016/S0003-2697(02)00206-3
language
English
LU publication?
yes
id
a5286926-4887-4c2c-b6bb-bb3486b0a947 (old id 892592)
date added to LUP
2008-01-18 15:59:58
date last changed
2017-01-01 04:30:29
@article{a5286926-4887-4c2c-b6bb-bb3486b0a947,
  abstract     = {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.},
  author       = {Rosen, M and Edfors-Lilja, I and Björnsson, Sven},
  issn         = {1096-0309},
  keyword      = {immobilization,membrane bound,solid phase,cationic detergent,glycosaminoglycan,proteoglycan,chondroitin sulfate,dermatan sulfate,heparan sulfate,epitopes,sequences,alcian blue,dot blot,transfer,blotting},
  language     = {eng},
  number       = {2},
  pages        = {210--222},
  publisher    = {Elsevier},
  series       = {Analytical Biochemistry},
  title        = {Quantitation of repetitive epitopes in glycosaminoglycans immobilized on hydrophobic membranes treated with cationic detergents},
  url          = {http://dx.doi.org/10.1016/S0003-2697(02)00206-3},
  volume       = {308},
  year         = {2002},
}