title: Development and evaluation of a fluorescence microplate assay for quantification of heparins and other sulfated carbohydrates.
abstract: Due to their complex composition, quantification of heparins is difficult. On the one hand there are many biological tests, which only indirectly detect effects of the antithrombin-binding material. On the other hand direct quantitative methods are available but they are often insensitive, challenging, time-consuming or expensive. The aim of this study was to develop a sensitive, rapid, simple as well as inexpensive direct quantification assay suitable for routine analysis. Based on Polymer-H, a novel heparin complexing, fluorescent labeled synthetic polymer (lambda((ex)) 320nm, lambda((em)) 510nm), a microplate assay was developed and optimized. The specificity of the assay was evaluated by structure-assay response relationships studies using structurally defined glucan sulfates, heparins, and other natural and synthetic sulfated carbohydrates. The fluorescence intensity of Polymer-H (7.5microg/ml) showed to be concentration-dependently amplified by heparins as well as by other sulfated carbohydrates. The best sensitivity, accuracy and linearity were observed in a range from 0.63 to 5.0microg/ml heparins. No differences in the fluorescence between various heparins were observed, so that only one calibration curve is needed. In addition, all types of carbohydrates with a degree of sulfation (DS)> approximately 1.2 and a M(r)>3000 can be quantified as well. By own calibration curves also other sulfated carbohydrates like fondaparinux or other glycosaminoglycans (DS>0.4) can be determined.
authors.affiliation: Pharmaceutical Institute, Christian-Albrechts-University, Kiel, Germany. sluehn@pharmazie.uni-kiel.de
authors.forename: Susanne
authors.initials: S
authors.lastname: Lühn
authors.forename: Thomas
authors.initials: T
authors.lastname: Schrader
authors.forename: Wei
authors.initials: W
authors.lastname: Sun
authors.forename: Susanne
authors.initials: S
authors.lastname: Alban
mesh_terms: D002138:Calibration; D004867:Equipment Design; D005453:Fluorescence; D005456:Fluorescent Dyes; D000077425:Fondaparinux; D006025:Glycosaminoglycans / Q000032:analysis*; D006493:Heparin / Q000032:analysis*; D006495:Heparin, Low-Molecular-Weight / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D008832:Microchemistry / Q000295:instrumentation / Q000592:standards; D015394:Molecular Structure; D008970:Molecular Weight; D009994:Osmolar Concentration; D011134:Polysaccharides / Q000032:analysis; D015203:Reproducibility of Results; D012965:Sodium Chloride / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D013696:Temperature; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D006025:Glycosaminoglycans; D006495:Heparin, Low-Molecular-Weight; D011134:Polysaccharides; D012965:Sodium Chloride; D006493:Heparin; D000077425:Fondaparinux