Biomedical Diagnostics Institute, National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland.
Talanta. 2010 Jun 15;81(4-5):1725-30. doi: 10.1016/j.talanta.2010.03.030. Epub 2010 Mar 25.
Fluorogenic assays have many potential advantages over traditional clot-based and chromogenic assays such as the absence of interference from a range of factor deficiencies as well as offering the possibility of assays in platelet rich plasma or whole blood. A fluorogenic anti-factor Xa (anti-FXa) assay has been developed for the determination of unfractionated heparin (UFH), low molecular weight heparins (LMWHs), namely enoxaparin and tinzaparin, and the synthetic heparinoid danaparoid, in commercial human pooled plasma. The assay was based on the complexation of heparin-spiked plasmas with exogenous FXa at a concentration of 4nM in the presence of 0.9microM of the fluorogenic substrate methylsulfonyl-D-cyclohexylalanyl-glycyl-arginine-7-amino-4-methylcoumarin acetate (Pefafluor FXa). Pooled plasma samples were spiked with concentrations of anticoagulants in the range 0-1.6U/ml. The assay was capable of the measurement of UFH and danaparoid in the range 0-1U/ml, and enoxaparin and tinzaparin in the range 0-0.8 and 0-0.6U/ml, respectively. Correlation coefficients generated by linear regression of the log/lin data analysis were between 0.93 and 0.96 for the anticoagulants tested. Assay percentage coefficients of variation were typically below 7%.
荧光测定法相对于传统的基于凝血和显色的测定法具有许多潜在的优势，例如不存在一系列因子缺乏的干扰，并且有可能在富含血小板的血浆或全血中进行测定。已经开发了一种荧光抗因子 Xa（抗-FXa）测定法，用于测定未分级肝素（UFH）、低分子量肝素（LMWH），即依诺肝素和亭扎肝素，以及合成肝素类似物达那肝素，在商业的人类混合血浆中。该测定法基于肝素缀合的血浆与外源性 FXa 在 4nM 浓度下在 0.9μM 的荧光底物甲基磺酰基-D-环己基丙氨酰基-甘氨酰-精氨酸-7-氨基-4-甲基香豆素乙酸酯（Pefafluor FXa）存在下的络合。混合血浆样品用抗凝剂的浓度在 0-1.6U/ml 范围内进行加标。该测定法能够测量 UFH 和达那肝素在 0-1U/ml 的范围内，以及依诺肝素和亭扎肝素在 0-0.8 和 0-0.6U/ml 的范围内。对数/线性数据分析的线性回归生成的相关系数对于所测试的抗凝剂在 0.93 到 0.96 之间。测定法的百分比变异系数通常低于 7%。
