Hafner G, Fickenscher K, Friesen H J, Rupprecht H J, Konheiser U, Ehrenthal W, Lotz J, Prellwitz W
Institute of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg-University Mainz, Germany.
Thromb Res. 1995 Jan 15;77(2):165-73. doi: 10.1016/0049-3848(95)91622-r.
A fully mechanized chromogenic substrate assay method for the rapid and specific determination of recombinant hirudin (r-hirudin) in citrated plasma on clinical chemistry analyzers (Hitachi 911 and Cobas Mira) is described. In a first step, 12 microliters sample volume is mixed with the chromogenic substrate. Due to the almost immediate action of hirudin the inhibitory reaction and the cleavage of the substrate is started simultaneously when bovine thrombin is added in excess. This excludes interferences by antithrombin III or heparin cofactor II. The change in absorbance/min is recorded at 405 nm. The measuring range is about 0.2-4.0 mg/l r-hirudin on both analyzers. Precision is characterized by intraassay coefficients of variation between 0.63% and 2.78% on the Hitachi 911 and 1.51% and 7.84% on the Cobas Mira, respectively and interassay coefficients of variation of 3.57% to 9.15% (Hitachi 911) and 3.72% to 12.99% (Cobas Mira) for the same r-hirudin plasma concentrations. The described determination of r-hirudin correlates well with an enzyme linked immunosorbent assay method for r-hirudin (Hitachi 911: r = 0.964, y = 0.978x + 0.038, n = 323; Cobas Mira: r = 0.964, y = 0.959x-0.003, n = 323).
本文描述了一种在临床化学分析仪（日立911和科霸米拉）上，用于快速、特异性测定枸橼酸盐血浆中重组水蛭素（r-水蛭素）的全机械化显色底物检测方法。第一步，将12微升样本体积与显色底物混合。由于水蛭素几乎立即发挥作用，当加入过量牛凝血酶时，抑制反应和底物裂解同时开始。这排除了抗凝血酶III或肝素辅因子II的干扰。在405nm处记录吸光度/分钟的变化。两台分析仪上r-水蛭素的测量范围均约为0.2 - 4.0mg/l。精密度的特征在于，日立911上的批内变异系数在0.63%至2.78%之间，科霸米拉上为1.51%至7.84%；对于相同r-水蛭素血浆浓度，批间变异系数在日立911上为3.57%至9.15%，在科霸米拉上为3.72%至12.99%。所描述的r-水蛭素测定方法与r-水蛭素的酶联免疫吸附测定方法相关性良好（日立911：r = 0.964，y = 0.978x + 0.038，n = 323；科霸米拉：r = 0.964，y = 0.959x - 0.003，n = 323）。
