A novel ELISA for the detection of active form of plasminogen activator inhibitor-1 based on a highly specific trapping agent
A novel ELISA for the detection of active form of plasminogen activator inhibitor-1 based on a highly specific trapping agent
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一种基于高度特异性捕获剂的新型 ELISA，用于检测活性形式的纤溶酶原激活剂抑制剂-1
DOI：
10.1016/j.aca.2018.12.005
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发表时间：
2019
影响因子：
6.2
通讯作者：
Huang M. D.
中科院分区：
文献类型：
--
作者：
Shang L;Xue G. P;Gong L. H;Zhang Y. X;Peng S. Z;Yuan C;Huang M. D.
纤溶酶原激活物抑制剂-1（Plasminogen activator inhibitor-1，派-1）是一种不稳定的分子，以四种不同的形式存在：活性形式、潜伏形式、切割形式和靶结合形式。尽管已有多种方法来测量总派-1，但活性形式的派-1抗原的测量仍然具有挑战性。在这里，我们开发了一种新的ELISA检测派-1的活性形式的基础上，高度特异性的派-1捕获剂，结合活性派-1具有高亲和力。我们还使用了一种高度稳定的派-1突变体作为测定校准品，以提高该方法的重现性。该ELISA具有同时测定派-1活性和抗原水平的优点。该方法的灵敏度为0.167 ng/ml，工作范围为0.195-25 ng/ml。批内和批间变异分别为6.7%和11.3%。加标标准品的平均回收率为102%。我们使用这种策略来测量健康供体血浆中的活性派-1水平，并且有一个有趣的观察结果：血浆在室温下储存6 h后派-1水平降低了一半。这一发现代表了血浆派-1样品中活性损失的首次观察结果，并且可以解释使用商业测定在人样品中观察到的派-1水平（0-100 ng/ml）的大的变化。
Plasminogen activator inhibitor-1 (PAI-1) is a labile molecule that exists in four different forms: active, latent, cleaved and target bound form. Although there have been many methods to measure the total PAI-1, the measurement of active form of PAI-1 antigen is still challenging. Here we developed a novel ELISA to detect the active form of PAI-1 based on a highly specific PAI-1 capturing agent which binds to active PAI-1 with high affinity. We also used a highly stable PAI-1 mutant as an assay calibrator to enhance the method's reproducibility. This ELISA has the advantage of measuring both the antigen level and activity of PAI-1 at the same time. The assay had a sensitivity of 0.167 ng/ml and a working range of 0.195–25 ng/ml. The intra- and inter-assay variations were 6.7% and 11.3% respectively. The mean recovery of spiked standard was 102%. We used this strategy to measure the active PAI-1 level in plasma of healthy donors, and had an interesting observation: the PAI-1 level reduced by half after plasma storage for 6 h at room temperature. This finding represents the first observation of activity loss in plasma PAI-1 samples, and may explain large variations in PAI-1 levels (0–100 ng/ml) observed in human samples using commercial assays.