Weijie Kong, Nonaka Toshiaki, Satoh Katsuya
Division of Cellular and Molecular Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki City 852-8501, Japan.
Department of Health Sciences, Unit of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki City 852-8523, Japan.
Diagnostics (Basel). 2024 Jul 15;14(14):1520. doi: 10.3390/diagnostics14141520.
Recently, the investigation of cerebrospinal fluid (CSF) biomarkers for diagnosing human prion diseases (HPD) has garnered significant attention. Reproducibility and accuracy are paramount in biomarker research, particularly in the measurement of total tau (T-tau) protein, which is a crucial diagnostic marker. Given the global impact of the coronavirus disease pandemic, the frequency of measuring this protein using one of the world's fully automated assays, chemiluminescent enzyme immunoassay (CLEA), has increased. At present, the diagnosis and monitoring of neurological diseases mainly rely on traditional methods, but their accuracy and responsiveness are limited. There is limited knowledge of the accuracy of CLEA in tau measurements. We aimed to measure T-tau protein using CLEA and to elucidate its merits and limitations.
We randomly selected 60 patients with rapidly progressive dementia, using ELISA and CLEA analysis of cerebrospinal fluid specimens. Additionally, we used Western blotting to detect the presence of 14-3-3 protein and employed real-time quaking-induced conversion (RT-QuIC) assays to analyze the same set of samples. Furthermore, we examined the correlation coefficient between ELISA and CLEA results in a subset of 60 samples. Moreover, using CLEA, we evaluated the diurnal reproducibility, storage stability, dilutability, and freeze-thaw effects in three selected samples.
In 172 patients, 172 samples were extracted, with each patient providing only one sample, and a total of 88 (35 men and 53 women) tested positive for HPD in the RT-QuIC assay. In contrast, all CSF samples from the remaining 84 patients without HPD (50 men and 34 women) tested negative in the RT-QuIC assay. Both ELISA and CLEA showed perfect sensitivity and specificity (100%) in measuring T-tau protein levels. In addition, ELISA and CLEA are similar in terms of measurement sensitivity and marginal effect of detection extrema. CLEA analysis exhibited instability for certain samples with T-tau protein levels exceeding 2000 pg/mL, leading to low reproducibility during dilution analysis.
Our findings indicate that CLEA outperforms ELISA in terms of diurnal reproducibility, storage stability, and freeze-thaw effects. However, ELISA demonstrated superior performance in the dilution assay. Therefore, it is imperative to develop innovative approaches for the dilution of biomarker samples for CLEA measurements during clinical trials.
最近，用于诊断人类朊病毒病（HPD）的脑脊液（CSF）生物标志物研究备受关注。可重复性和准确性在生物标志物研究中至关重要，尤其是在总tau（T-tau）蛋白的测量方面，T-tau蛋白是一种关键的诊断标志物。鉴于冠状病毒病大流行的全球影响，使用全球全自动检测方法之一化学发光酶免疫分析（CLEA）测量这种蛋白的频率有所增加。目前，神经疾病的诊断和监测主要依赖传统方法，但其准确性和反应性有限。关于CLEA在tau测量中的准确性的了解有限。我们旨在使用CLEA测量T-tau蛋白，并阐明其优点和局限性。
我们随机选择60例快速进展性痴呆患者，对脑脊液标本进行酶联免疫吸附测定（ELISA）和CLEA分析。此外，我们使用蛋白质印迹法检测14-3-3蛋白的存在，并采用实时震颤诱导转化（RT-QuIC）测定法分析同一组样本。此外，我们检查了60个样本子集中ELISA和CLEA结果之间的相关系数。此外，我们使用CLEA评估了三个选定样本的昼夜可重复性、储存稳定性、稀释性和冻融效应。
在172例患者中，提取了172个样本，每位患者仅提供一个样本，在RT-QuIC测定中，共有88例（35名男性和53名女性）HPD检测呈阳性。相比之下，其余84例无HPD的患者（50名男性和34名女性）的所有脑脊液样本在RT-QuIC测定中均呈阴性。ELISA和CLEA在测量T-tau蛋白水平方面均显示出完美的敏感性和特异性（100%）。此外，ELISA和CLEA在测量敏感性和检测极值的边际效应方面相似。对于某些T-tau蛋白水平超过2000 pg/mL的样本，CLEA分析表现出不稳定性，导致稀释过程中的可重复性较低。
我们的研究结果表明，在昼夜可重复性、储存稳定性和冻融效应方面，CLEA优于ELISA。然而，ELISA在稀释测定中表现出更好的性能。因此，在临床试验期间，必须开发创新方法来稀释用于CLEA测量的生物标志物样本。
