Philippou H, Adami A, Amersey R A, Stubbs P J, Lane D A
Department of Haematology, Charing Cross and Westminster Medical School, London, UK.
Blood. 1997 Feb 1;89(3):767-75.
We report the development of an enzyme-linked immunosorbent assay (ELISA) that is specific for factor VIIa (FVIIa). This assay uses a neoantigen specific capture antibody directed to the amino acid peptide sequence N terminal to the FVII cleavage activation site. The antibody exhibits approximately 3,000-fold greater reactivity to FVIIa than FVII on a molar basis. Experiments using plasma with added (exogenous) human FVIIa gave quantitative recovery in the ELISA over a range of 0.20 to 3.2 ng/mL of FVIIa. The intra- and inter-assay coefficient of variation (CVs) of the ELISA are 4.5% and 9.8%, respectively. The ELISA shows excellent correlation (r = .99) with a functional assay (using recombinant soluble tissue factor) in detecting FVIIa added to plasma over the range 0.05 to 18.0 ng/mL. However, a major discrepancy exists between the two assays when normal endogenous plasma concentrations of FVIIa are measured. Using normal plasma (n = 14) the functional assay reported 3.10 +/- 0.30 ng/mL (mean +/- SE) whereas only 0.025 +/- 0.010 ng/mL was detected in the same samples by the immunoassay. Patients (n = 43) presenting with acute coronary syndromes (myocardial infarction and unstable angina) exhibited elevations (P < .05) in immunologically detected FVIIa, 0.093 +/- 0.013 ng/mL (mean +/- SE) compared to patient controls (n = 20) contemporaneously admitted with noncardiac chest pain, 0.048 +/- 0.007 ng/mL (mean +/- SE). These elevations in the acute coronary syndromes were accompanied by increased (P < .05) and correlating prothrombin fragment F1 + 2 levels (Spearman correlation coefficient rs = .4, P < .01), demonstrating that thrombin generation is certainly associated with, and may even be caused by, extrinsic pathway activation.
我们报告了一种针对活化凝血因子VII（FVIIa）的酶联免疫吸附测定（ELISA）的研发情况。该测定法使用一种针对FVII裂解激活位点N端氨基酸肽序列的新抗原特异性捕获抗体。在摩尔基础上，该抗体对FVIIa的反应性比对FVII高约3000倍。使用添加了（外源性）人FVIIa的血浆进行的实验在0.20至3.2 ng/mL的FVIIa范围内，ELISA检测结果呈定量回收。ELISA的批内和批间变异系数（CVs）分别为4.5%和9.8%。在检测添加到血浆中的FVIIa时，ELISA在0.05至18.0 ng/mL范围内与功能测定法（使用重组可溶性组织因子）显示出极好的相关性（r = 0.99）。然而，在测量正常内源性血浆FVIIa浓度时，两种测定法之间存在重大差异。使用正常血浆（n = 14）时，功能测定法报告的浓度为3.10 +/- 0.30 ng/mL（平均值 +/- 标准误），而免疫测定法在相同样本中仅检测到0.025 +/- 0.010 ng/mL。患有急性冠状动脉综合征（心肌梗死和不稳定型心绞痛）的患者（n = 43）免疫检测到的FVIIa水平升高（P < 0.05），为0.093 +/- 0.013 ng/mL（平均值 +/- 标准误），而同期因非心脏性胸痛入院的患者对照组（n = 20）为0.048 +/- 0.007 ng/mL（平均值 +/- 标准误）。急性冠状动脉综合征患者FVIIa水平的升高伴随着凝血酶原片段F1 + 2水平的升高（P < 0.05）且二者相关（Spearman相关系数rs = 0.4，P < 0.01），表明凝血酶的生成肯定与外源性途径激活相关，甚至可能由其引起。
