P102 Development of a Novel Assay for the Detection of Active Neutrophil Elastase in Patients with Chronic Obstructive Pulmonary Disease
P102 Development of a Novel Assay for the Detection of Active Neutrophil Elastase in Patients with Chronic Obstructive Pulmonary Disease
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P102 开发一种检测慢性阻塞性肺病患者活性中性粒细胞弹性蛋白酶的新方法
DOI：
10.1136/thoraxjnl-2015-207770.239
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发表时间：
2015
期刊：
影响因子：
10
通讯作者：
Moffitt K
中科院分区：
文献类型：
--
作者：
Moffitt K
中性粒细胞弹性蛋白酶（NE）是一种感染和炎症的生物标志物，与包括慢性阻塞性肺疾病（COPD）在内的几种呼吸系统疾病的严重程度相关。然而，由于缺乏可靠和稳健的方法，它在生物样品中的检测和定量受到了困扰。使用显色或荧光底物的标准分析在添加到含有多种具有水解底物能力的蛋白水解酶和水解酶的复杂临床样品时不具有特异性，从而导致对目标蛋白酶的高估。此外，ELISA系统测量总蛋白酶水平，它可以是潜伏的，活性的和蛋白酶抑制剂复合物的混合物。因此，我们开发了一种新的免疫测定方法（ProteaseTag™Active NE immunoassay），该方法具有选择性和特异性，可用于捕获COPD患者痰液和支气管肺泡灌洗（BAL）中的活性vene。本研究的目的是临床验证ProteaseTag™Active NE Ultra Immunoassay检测COPD患者痰液中的NE。从10例COPD患者（M = 6, F = 4； 73±6年）的稳定期和加重期中采集20份匹配的痰液样本。使用ProteaseTag™Active NE Ultra Immunoassay和基于荧光底物的动力学活性测定法检测样品的NE活性。两种方法均检测到大多数患者（n = 7）在急性加重期（平均= 217.2µg/ml±296.6）与稳定期（平均= 92.37µg/ml±259.8）相比，NE水平升高。然而，统计分析并没有显示这种差异是显著的（p = 0.07, ProteaseTag™Active NE Ultra Immunoassay; p = 0.06 kinetic assay），这很可能是由于研究数量少。两种检测类型间相关性极显著（p≤0.0001,r = 0.996）。NE作为临床试验的主要疗效终点或作为临床炎症标志物，由于缺乏一种强大且简单易用的检测方法而受到阻碍。ProteaseTag™Active NE免疫测定法专门测量临床样品中的activene，使用快速简便（<3小时），不依赖于动力学读数。ProteaseTag™技术目前正在转移到一个横向流动设备，用于护理点。
Neutrophil elastase (NE), a biomarker of infection and inflammation, correlates with the severity of several respiratory diseases including chronic obstructive pulmonary disease (COPD). However, it’s detection and quantification in biological samples is confounded by a lack of reliable and robust methodologies. Standard assays using chromogenic or fluorogenic substrates are not specific when added to complex clinical samples containing multiple proteolytic and hydrolytic enzymes which have the ability to hydrolyse the substrate, thereby resulting in an over-estimation of the target protease. Furthermore, ELISA systems measure total protease levels which can be a mixture of latent, active and protease-inhibitor complexes. Therefore, we have developed a novel immunoassay (ProteaseTag™ Active NE Immunoassay) which is selective and specific for the capture ofactiveNE in sputum and Bronchoalveolar Lavage (BAL) in patients with COPD.The objective of this study was to clinically validate ProteaseTag™ Active NE Ultra Immunoassay for the detection of NE in sputum from COPD patients.20 matched sputum sol samples were collected from 10 COPD patients (M = 6, F = 4; 73 ± 6 years) during stable and exacerbation phases. Samples were assayed for NE activity utilising both ProteaseTag™ Active NE Ultra Immunoassay and a fluorogenic substrate-based kinetic activity assay.Both assays detected elevated levels of NE in the majority of patients (n = 7) during an exacerbation (mean = 217.2 µg/ml ±296.6) compared to their stable phase (mean = 92.37 µg/ml ±259.8). However, statistical analysis did not show this difference to be significant (p = 0.07, ProteaseTag™ Active NE Ultra Immunoassay; p = 0.06 kinetic assay), which is highly likely to be due to the low study number. A highly significant correlation was found between the 2 assay types (p ≤ 0.0001, r = 0.996).NE as a primary efficacy endpoint in clinical trials or as a marker of inflammation within the clinic has been hampered by the lack of a robust and simple to use assay. ProteaseTag™ Active NE Immunoassay specifically measures onlyactiveNE in clinical samples, is quick and easy to use (<3 h) and has no dependency on a kinetic readout. ProteaseTag™ technology is currently being transferred to a lateral flow device for use at Point of Care.