The close correlation between Tau pathology and Alzheimer's disease (AD) progression makes this protein a suitable biomarker for diagnosis and monitoring of the disorder evolution. However, the use of Tau in diagnostics has been hampered, as it currently requires collection of cerebrospinal fluid (CSF), which is an invasive clinical procedure. Although measuring Tau-levels in blood plasma would be favorable, the concentrations are below the detection limit of a conventional ELISA. In this work, we developed a digital ELISA for the quantification of attomolar protein Tau concentrations in both buffer and biological samples. Individual Tau molecules were first captured on the surface of magnetic particles using in-house developed antibodies and subsequently isolated into the femtoliter-sized wells of a 2 × 2 mm2 microwell array. Combination of high-affinity antibodies, optimal assay conditions and a digital quantification approach resulted in a 24 ± 7 aM limit of detection (LOD) in buffer samples. Additionally, a dynamic range of 6 orders of magnitude was achieved by combining the digital readout with an analogue approach, allowing quantification from attomolar to picomolar levels of Tau using the same platform. This proves the compatibility of the presented assay with the wide range of Tau concentrations encountered in different biological samples. Next, the developed digital assay was applied to detect total Tau levels in spiked blood plasma. A similar LOD (55 ± 29 aM) was obtained compared to the buffer samples, which was 5000-fold more sensitive than commercially available ELISAs and even outperformed previously reported digital assays with 10-fold increase in sensitivity. Finally, the performance of the developed digital ELISA was assessed by quantifying protein Tau in three clinical CSF samples. Here, a high correlation (i.e. Pearson coefficient of 0.99) was found between the measured percentage of active particles and the reference protein Tau values. The presented digital ELISA technology has great capacity in unlocking the potential of Tau as biomarker for early AD diagnosis.

中文翻译：

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数字 ELISA 用于量化生物样品中阿尔茨海默病生物标志物蛋白 Tau 的阿摩尔浓度

Tau 病理学与阿尔茨海默病 (AD) 进展之间的密切相关性使该蛋白质成为诊断和监测疾病演变的合适生物标志物。然而，Tau 在诊断中的使用受到了阻碍，因为它目前需要收集脑脊液 (CSF)，这是一种侵入性的临床程序。尽管测量血浆中的 Tau 水平是有利的，但其浓度低于传统 ELISA 的检测限。在这项工作中，我们开发了一种数字 ELISA，用于量化缓冲液和生物样品中的 attomolar 蛋白 Tau 浓度。首先使用内部开发的抗体在磁性颗粒表面捕获单个 Tau 分子，然后将其分离到 2 × 2 mm2 微孔阵列的飞升大小的孔中。高亲和力抗体、最佳检测条件和数字量化方法的组合导致缓冲样品中的检测限 (LOD) 为 24 ± 7 aM。此外，通过将数字读数与模拟方法相结合，实现了 6 个数量级的动态范围，允许使用同一平台从阿托摩尔到皮摩尔水平的 Tau 进行量化。这证明了所提出的测定与不同生物样品中遇到的各种 Tau 浓度的兼容性。接下来，开发的数字化验被应用于检测加标血浆中的总 Tau 水平。与缓冲液样品相比，获得了类似的 LOD (55 ± 29 aM)，其灵敏度比市售的 ELISA 方法高 5000 倍，甚至优于之前报道的数字检测方法，灵敏度提高了 10 倍。最后，通过量化三个临床脑脊液样本中的蛋白质 Tau 来评估开发的数字 ELISA 的性能。在此，发现活性颗粒的测量百分比与参考蛋白质 Tau 值之间存在高度相关性（即 Pearson 系数为 0.99）。所提出的数字 ELISA 技术在释放 Tau 作为早期 AD 诊断生物标志物的潜力方面具有巨大的能力。99) 被发现在活性颗粒的测量百分比和参考蛋白质 Tau 值之间。所提出的数字 ELISA 技术在释放 Tau 作为早期 AD 诊断生物标志物的潜力方面具有巨大的能力。99) 被发现在活性颗粒的测量百分比和参考蛋白质 Tau 值之间。所提出的数字 ELISA 技术在释放 Tau 作为早期 AD 诊断生物标志物的潜力方面具有巨大的能力。