A solid-phase assay for quantitative analysis of sulfated glycosaminoglycans at the nanogram level. Application to tissue samples.
A sensitive and accurate solid-phase methodology for the quantitative analysis of glycosaminoglycans is described. Chondroitin-4-sulfate (CSA) was labelled with biotin hydrazide after the reaction of its carboxyl groups with it in the presence of carbodiimide. Polystyrene plates modified with sequential reaction with glutaraldehyde (GH) and spermine to possess amino groups were used to immobilize electrostatically the biotin labelled CSA. Exogenously added sulfated glycosaminoglycans (GAGS) [variously sulfated chondroitin sulfates and heparan sulfate (HS)] were found to compete to this immobilization in a concentration dependent mode, within a concentration range from 10 up to 300 ng/ml. Glycosaminoglycan-derived oligosaccharides competed to a degree similar to that of intact molecules. Hyaluronan (HA) and keratan sulfate (KS) did not compete the immobilization. The procedure was applied for the rapid and reproducible determination of the sulfated glycosaminoglycans in proteinase digests of small tissue samples or cell cultures with high sensitivity and accuracy.
Vynios
D H
DH
Laboratory of Biochemistry, Department of Chemistry, University of Patras, Greece. vynios@chemistry.upatras.gr
Faraos
A
A
Spyracopoulou
G
G
Aletras
A J
AJ
Tsiganos
C P
CP
D000465:Algorithms; D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry; D002234:Carbodiimides; D002478:Cells, Cultured / Q000737:chemistry; D002809:Chondroitin Sulfates / Q000737:chemistry; D010450:Endopeptidases; D006025:Glycosaminoglycans / Q000032:analysis*; D006820:Hyaluronic Acid / Q000737:chemistry; D007632:Keratan Sulfate / Q000737:chemistry; D008832:Microchemistry / Q000379:methods; D009844:Oligosaccharides / Q000737:chemistry; D015203:Reproducibility of Results; D012756:Sheep; D013431:Sulfates / Q000032:analysis; D013552:Swine
D002234:Carbodiimides; D006025:Glycosaminoglycans; D009844:Oligosaccharides; D013431:Sulfates; C053311:biotin hydrazide; D001710:Biotin; D006820:Hyaluronic Acid; D002809:Chondroitin Sulfates; D007632:Keratan Sulfate; D010450:Endopeptidases