Novel Multiplex Assay for Biomarkers of Alzheimer's Disease

描述(申请人提供)：阿尔茨海默病(AD)是痴呆症最常见的形式，是一种复杂的疾病，很难诊断和治疗。目前，还没有现成的生物标记物测试小组来帮助诊断或监测疾病进展。虽然脑脊液中生物标记物的分析提供了有用的信息，但血液中生物标记物的有效性还没有得到很好的记录。该项目的目标是开发一种用于阿尔茨海默病患者血液样本的多重生物标记物测试。在这笔赠款的第一阶段，我们将论证一种新的快速多重检测AD血清/血浆生物标记物的可行性。我们开发了一种专利方法，使用实时聚合酶链式反应(PCR)技术以非常高的灵敏度和重复性以多重方式测量蛋白质浓度。该方法被称为多路实时免疫-聚合酶链式反应(MRI-PCR)，由夹心免疫分析组成，其中检测抗体被不同的DNA序列标记。然后使用实时荧光聚合酶链式反应对所有DNA标记进行并行量化，从而揭示生物样本中同源蛋白质的数量。初步数据表明，与标准的酶联免疫吸附试验相比，使用核磁共振-聚合酶链式反应检测的多重细胞因子检测的灵敏度提高了~2个数量级，动态范围增加了2个数量级，标准偏差非常低(CV&lt；10%)，并且具有很高的特异性。在第一阶段的研究中，将准备一组试剂来检测15个人类AD生物标记物，并在MRI-PCR分析中进行评估。然后将目标蛋白添加到正常的人血清中，以在生理样本中验证该分析。要评估的参数包括灵敏度、动态范围、准确度、精密度、剂量-反应线性和特异性。将进行一项小型试点研究，以评估该测试在AD患者和年龄匹配的正常对照组(n=10-20个样本)的血清样本上的性能。在第二阶段，该分析将在一项大规模的临床研究中进一步得到验证，以证明该技术在AD的诊断、预后和治疗监测中的价值。这些研究的结果将导致一种新的AD检测方法，它比脑脊液分析侵入性更小，并将为临床医生和研究人员提供有价值的信息。公共卫生相关性：该项目将开发一种新的检测方法，以帮助诊断、预后和监测阿尔茨海默病的治疗。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD), the most common form of dementia, is a complex disorder that is difficult to diagnose and treat. Currently, there is no readily available biomarker test panel to aid diagnosis or monitor disease progression. Although analysis of biomarkers in cerebrospinal fluid (CSF) has provided useful information, the validation of biomarkers in blood has not been as well-documented. The goal of this project is to develop a multiplex biomarker test for blood samples from Alzheimer's patients. In Phase I of this grant, we will demonstrate the feasibility of a novel rapid multiplex assay for serum/plasma biomarkers of AD. We have developed a proprietary method for using the real-time-polymerase chain reaction (PCR) technology to measure protein concentrations in a multiplexed fashion with very high sensitivity and reproducibility. The method, Multiplexed Real-Time-Immuno-PCR (MRI-PCR), consists of a sandwich immuno-assay in which the detection antibodies are labeled with different DNA sequences. Real-time PCR is then used to quantify all of the DNA labels in parallel, thus revealing the quantities of the cognate proteins in the biological samples. Preliminary data have demonstrated that multiplexed cytokine measurements employing MRI-PCR assays provides ~2 orders of magnitude more sensitive detection and >2 orders of magnitude increased dynamic range in comparison to standard ELISA assays, along with very low standard deviations (CV <10%) and high specificity. In Phase I studies, a panel of reagents to detect 15 human biomarkers of AD will be prepared and evaluated in the MRI-PCR assay. The target proteins will then be spiked into normal human serum to validate the assay in a physiological sample. Parameters to be evaluated include sensitivity, dynamic range, accuracy, precision, dose-response linearity, and specificity. A small pilot study will be conducted to evaluate the performance of the test on serum samples from AD patients as well as age-matched normal controls (n=10-20 samples). In Phase II, the assay will be further validated in a large scale clinical study to demonstrate the value of this technology in the diagnosis, prognosis and monitoring of therapy for AD. The results of these studies will lead to a new test for AD that is less invasive than CSF analysis and will provide valuable information to both clinicians as well as researchers. PUBLIC HEALTH RELEVANCE: This project will develop a novel assay to assist in the diagnosis, prognosis, and monitoring of therapy for Alzheimer's disease.