Biomedical Diagnostics Institute, National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland.
Anal Bioanal Chem. 2011 Jan;399(2):691-700. doi: 10.1007/s00216-010-4314-3. Epub 2010 Oct 24.
Low molecular weight heparins (LMWHs) are recognised as the preferred anticoagulants in the prevention and treatment of venous thromboembolism. Anti-Factor Xa (anti-FXa) levels are used to monitor the anticoagulant effect of LMWHs and such assays are routinely employed in hospital diagnostic laboratories. In this study, a fluorogenic anti-FXa assay was developed using a commercially available fluorogenic substrate with an attached 6-amino-1-naphthalene-sulfonamide (ANSN) fluorophore and was used for the determination of two LMWHs, enoxaparin and tinzaparin and the heparinoid, danaparoid. The assay was based on the complexation of heparinised plasma with 100 nM exogenous FXa and 25 μM of the fluorogenic substrate Mes-D-LGR-ANSN (C(2)H(5))(2) (SN-7). The assay was tested with pooled plasma samples spiked with anticoagulant concentrations in the range 0-1.6 U mL(-1). The statistically sensitive assay range was 0-0.4 U mL(-1) for enoxaparin and tinzaparin and 0-0.2 U mL(-1) for danaparoid, with assay variation typically below 10.5%. This assay was then compared with a previously published fluorogenic anti-FXa assay developed with the peptide substrate, methylsulfonyl-D: -cyclohexylalanyl-glycyl-arginine-7-amino-4-methylcoumarin acetate (Pefafluor FXa). Both assays were compared in terms of fluorescence intensity, lag times and sensitivity to anticoagulants.
低分子量肝素（LMWHs）被认为是预防和治疗静脉血栓栓塞症的首选抗凝剂。抗因子 Xa（抗 FXa）水平用于监测 LMWHs 的抗凝效果，并且此类测定通常在医院诊断实验室中进行。在这项研究中，使用市售的带有附着的 6-氨基-1-萘磺酰胺（ANSN）荧光团的荧光底物开发了荧光抗 FXa 测定法，并用于测定两种 LMWHs，依诺肝素和亭扎肝素以及肝素类药物达那肝素。该测定法基于肝素化血浆与 100 nM 外源性 FXa 和 25 μM 荧光底物 Mes-D-LGR-ANSN（C（2）H（5））（2）（SN-7）的络合。该测定法使用浓度范围为 0-1.6 U mL（-1）的抗凝剂的混合血浆样品进行了测试。对于依诺肝素和亭扎肝素，统计上灵敏的测定范围为 0-0.4 U mL（-1），对于达那肝素，测定范围为 0-0.2 U mL（-1），测定变异通常低于 10.5％。然后，将该测定法与先前使用肽底物（甲基磺酰基-D：-环己基丙氨酸-甘氨酸-精氨酸-7-氨基-4-甲基香豆素乙酸酯（Pefafluor FXa））开发的荧光抗 FXa 测定法进行了比较。就荧光强度，滞后时间和对抗凝剂的敏感性而言，比较了两种测定法。
