Highly sensitive immunoassay for determination of biomarkers for neurodegenerative diseases

摘要 该提案的长期目标是进一步开发光化学信号放大方法 （PSAM）用于增加常规酶联免疫吸附测定（ELISA）的灵敏度， 用于确定各种神经退行性疾病（例如阿尔茨海默病（AD））的生物标志物。 免疫酶法如ELISA广泛用于生物学和医学中的药物筛选， 检测病毒（包括冠状病毒）、细菌和生物标志物，特别是细胞因子和白细胞介素， 癌症和免疫紊乱，以及神经变性疾病的生物标记物。他们经常使用 在执行大量测试的手动、半自动和自动系统中。但在 在许多情况下，ELISA的灵敏度不足。 早期诊断AD的一些关键生物标志物包括三种脑脊液（CSF）生物标志物： 淀粉样蛋白β-42（AAP 14 - 42），其导致寡聚斑块、总tau（t-tau）和磷酸化tau的形成 （p-tau）（其增加细胞内神经元缠结（NFT））。迄今为止，没有敏感和成本- 定量这三种蛋白质的有效方法，阻碍了诊断和进展 监测AD。 现有的高灵敏度方法繁琐且昂贵。因此， 需要一个通用ELISA平台来检测低水平的Δ tau-42、t-tau、p-tau和其他生物标志物， 各种神经退行性疾病，既便宜又简单，不需要专门的 实验室设备.在我们的第一阶段项目中，我们建议进一步开发一种非常敏感和廉价的 用于检测各种神经退行性疾病的生物标志物的免疫测定平台。拟议 ELISA + PSAM技术由两个步骤组成：（1）常规辣根过氧化物酶（HRP）介导的 一种酶联免疫吸附试验（ELISA），其中使用一种普通的显色HRP底物，和（2）一种底物溶液，含有 用可见光照射酶促反应的产物。样品的照明导致DAP- 催化DAP浓度的急剧增加（自催化光化学反应）。 第一阶段的具体目标是： 1.探索影响ELISA + PSAM检测法用于检测A142、t-tau的性能的所有变量 和p-tau，并优化测定的性能。制定一个普遍的办法， ELISA至ELISA + PSAM测定。 2.通过使用最多3种方法，验证优化ELISA + PSAM测定的通用方法 用于每种分析物的来自不同制造商的市售ELISA试剂盒。这将包括决心 检测的灵敏度和重现性/精密度。

Abstract The long-term objective of this proposal is to further develop a photochemical signal amplification method (PSAM) for increasing the sensitivity of conventional enzyme-linked immunosorbent assays (ELISA) for determination of biomarkers for various neurodegenerative diseases, such as Alzheimer’s Disease (AD). Immunoenzymatic methods such as ELISA are widely used in biology and medicine for drug screening, for detecting viruses (including coronaviruses), bacteria, and biomarkers, particularly cytokines and interleukins for cancer and immunological disorders, and biomarkers for neurodegenerative diseases. They are routinely used in manual, semi-automated, and automated systems where high volumes of tests are performed. However, in many cases the sensitivity of ELISA is inadequate. Some of the key biomarkers for early diagnosis of AD include three cerebrospinal fluid (CSF) biomarkers: amyloid ß-42 (Aß-42), which causes formation of oligomeric plaques, total tau (t-tau), and phosphorylated tau (p-tau) (which increases intracellular neurofibrillary tangles (NFTs). To date, there is no sensitive and cost- effective method for the quantification of these three proteins, hindering the diagnosis and progression monitoring of AD. Existing highly sensitive methods are cumbersome and expensive. Therefore, there is an increased necessity for a common ELISA platform to detect low levels of Aß-42, t-tau, p-tau and other biomarkers for various neurodegenerative diseases that is both inexpensive and simple enough to not require specialized laboratory equipment. In our Phase I project, we propose to further develop a very sensitive and inexpensive immunoassay platform for detection of biomarkers for various neurodegenerative diseases. The proposed technology, ELISA + PSAM, consists of two steps: (1) a conventional horseradish peroxidase (HRP)-mediated assay (ELISA) in which a common chromogenic HRP substrate, is used and (2) a substrate solution containing the product of the enzymatic reaction is irradiated by visible light. Illumination of the sample leads to a DAP- catalyzed drastic increase in DAP concentration (autocatalytic photochemical reaction). The Specific aims of Phase I are: 1. To explore all variables affecting the performance of ELISA + PSAM assays for detection of Aß-42, t-tau and p-tau and optimize the performance of the assays. To develop a universal approach for transitioning from ELISA to ELISA + PSAM assays. 2. To validate the universal approach for optimization of ELISA + PSAM assays by using up to 3 commercially available ELISA kits from different manufacturers for each analyte. This will include determination of sensitivity and reproducibility/precision of the assays.