Development of an enzyme-linked immunosorbent assay (ELISA)-like fluorescence assay to investigate the interactions of glycosaminoglycans to cells

Development of an enzyme-linked immunosorbent assay (ELISA)-like fluorescence assay to investigate the interactions of glycosaminoglycans to cells

Autor Boucas, Rodrigo Ippolito Autor UNIFESP Google Scholar
Trindade, Edvaldo S. Autor UNIFESP Google Scholar
Tersariol, Ivarne L. S. Autor UNIFESP Google Scholar
Dietrich, Carl P. Autor UNIFESP Google Scholar
Nader, Helena B. Autor UNIFESP Google Scholar
Instituição Universidade Federal de São Paulo (UNIFESP)
Univ Fed Parana
Univ Mogi das Cruzes
Resumo Sulfated glycosaminoglycans were labeled with biotin to study their interaction with cells in culture. Thus, heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate were labeled using biotin-hydrazide, under different conditions. the structural characteristics of the biotinylated products were determined by chemical (molar ratios of hexosamine, uronic acid, sulfate and biotin) and enzymatic methods (susceptibility to degradation by chondroitinases and heparitinases). the binding of biotinylated glycosaminoglycans was investigated both in endothelial and smooth muscle cells in culture, using a novel time resolved fluorometric method based on interaction of europium-labeled streptavidin with the biotin covalently linked to the compounds. the interactions of glycosaminoglycans were saturable and number of binding sites could be obtained for each individual compound. the apparent dissociation constant varied among the different glycosaminoglycans and between the two cell lines, the interactions of the biotinylated glycosaminoglycans with the cells were also evaluated using confocal microscopy. We propose a convenient and reliable method for the preparation of biotinylated glycosaminoglycans, as well as a sensitive non-competitive fluorescence-based assay for studies of the interactions and binding of these compounds to cells in culture. (c) 2008 Elsevier B.V. All rights reserved.
Assunto glycosaminoglycans
biotinylation procedures
fluorescence
endothelial cells
smooth muscle cells
Idioma Inglês
Data 2008-06-23
Publicado em Analytica Chimica Acta. Amsterdam: Elsevier B.V., v. 618, n. 2, p. 218-226, 2008.
ISSN 0003-2670 (Sherpa/Romeo, fator de impacto)
Editor Elsevier B.V.
Extensão 218-226
Fonte http://dx.doi.org/10.1016/j.aca.2008.04.059
Direito de acesso Acesso restrito
Tipo Artigo
Web of Science WOS:000257004400011
URI http://repositorio.unifesp.br/handle/11600/30737

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