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.
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