Immobilization of Proteinase K for urine pretreatment to improve diagnostic accuracy of active tuberculosis.
Immobilization of Proteinase K for urine pretreatment to improve diagnostic accuracy of active tuberculosis.
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DOI：
10.1371/journal.pone.0257615
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发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
Chatterjee D
中科院分区：
文献类型：
--
作者：
Panraksa Y;Amin AG;Graham B;Henry CS;Chatterjee D
世界卫生组织（世卫组织）呼吁开发一种快速、基于生物标志物的无痰检测方法，能够在护理点检测所有形式的结核病（TB），以便立即开始治疗。脂阿拉伯甘露聚糖（LAM）是唯一一种世卫组织认可的结核病生物标志物，可以在尿液中检测到，尿液是一种容易收集的样本基质。为了获得最佳灵敏度，我们和其他人已经表明，需要某种形式的样本预处理来去除患者尿液样本中的背景。许多系统是基于纸张的，通常用于资源有限的环境。我们目前的工作提出了一个这样的样品预处理，蛋白酶K（ProK）固定在纸上（IPK），并测试其性能相比，标准蛋白酶K（SPK）的处理，涉及添加和失活在高温下进行捕获ELISA之前。本研究建立了一种简便、经济的尿标本前处理方法。通过使用Whatman 1号纸和通过最小化用于在ELISA之前预处理临床样品的ProK（一种昂贵但必需的试剂）的浓度，实现了所提出的预处理条的简化和成本降低。为了测试IPK的适用性，在室温下用400 μg/mL的ProK预处理30分钟后，对加标LAM的尿液或临床样品进行捕获ELISA。对IPK的最佳条件和稳定性进行了检测，并对一组25份先前分析的存档临床尿液样本（已知TB和HIV状态）进行了验证。IPK和SPK处理样本的结果一致，表明目前正在开发的尿液LAM检测有可能达到成人和儿童患者，无论HIV状态或感染部位如何，并促进全球结核病控制，以改善检测性能并最终改善治疗结果。
The World Health Organization (WHO) calls for the development of a rapid, biomarker-based, non-sputum test capable of detecting all forms of tuberculosis (TB) at the point-of-care to enable immediate treatment initiation. Lipoarabinomannan (LAM) is the only WHO-endorsed TB biomarker that can be detected in urine, an easily collected sample matrix. For obtaining optimal sensitivity, we and others have shown that some form of sample pretreatment is necessary to remove background from patient urine samples. A number of systems are paper-based often destined for resource limited settings. Our current work presents incorporation of one such sample pretreatment, proteinase K (ProK) immobilized on paper (IPK) and test its performance in comparison to standard proteinase K (SPK) treatment that involves addition and deactivation at high temperature prior to performing a capture ELISA. Herein, a simple and economical method was developed for using ProK immobilized strips to pretreat urine samples. Simplification and cost reduction of the proposed pretreatment strip were achieved by using Whatman no.1 paper and by minimizing the concentration of ProK (an expensive but necessary reagent) used to pretreat the clinical samples prior to ELISA. To test the applicability of IPK, capture ELISA was carried out on either LAM-spiked urine or the clinical samples after pretreatment with ProK at 400 μg/mL for 30 minutes at room temperature. The optimal conditions and stability of the IPK were tested and validation was performed on a set of 25 previously analyzed archived clinical urine samples with known TB and HIV status. The results of IPK and SPK treated samples were in agreement showing that the urine LAM test currently under development has the potential to reach adult and pediatric patients regardless of HIV status or site of infection, and to facilitate global TB control to improve assay performance and ultimately treatment outcomes.
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影响因子：
3.2
作者：
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通讯作者：
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影响因子：
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作者：
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DOI：
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发表时间：
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期刊：
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影响因子：
--
作者：
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通讯作者：
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影响因子：
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作者：
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影响因子：
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