Highly sensitive immunoassay for determination of biomarkers for neurodegenerative diseases

摘要 这项提议的长期目标是进一步开发一种光化学信号放大方法 (PSAM)用于提高传统的酶联免疫吸附试验(ELISA)对 各种神经退行性疾病的生物标志物的测定，如阿尔茨海默病(AD)。 免疫酶方法，如酶联免疫吸附试验，在生物学和医学中被广泛应用于药物筛选， 检测病毒(包括冠状病毒)、细菌和生物标记物，特别是细胞因子和白介素类 癌症和免疫紊乱，以及神经退行性疾病的生物标记物。它们是日常使用的 在执行大量测试的手动、半自动和自动化系统中。但是，在 很多情况下，酶联免疫吸附试验的敏感性不足。 早期诊断AD的一些关键生物标记物包括三个脑脊液(CSF)生物标记物： 淀粉样蛋白-42(A？-42)，可导致低聚斑块、总tau(t-tau)和磷酸化tau的形成 (P-tau)(增加细胞内神经原纤维缠结(NFTs)。到目前为止，还没有敏感和成本- 这三种蛋白定量的有效方法，阻碍了诊断和进展 AD的监测。 现有的高度敏感的方法既繁琐又昂贵。因此，有一个增加了 需要一个通用的酶联免疫吸附试验平台来检测低水平的A？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生物标志物的免疫分析平台。建议数 该技术由两个步骤组成：(1)传统的辣根过氧化物酶(HRP)介导的 检测(ELISA)，其中使用共同的显色HRP底物，和(2)含有 酶反应的产物用可见光照射。样品的光照导致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.探讨影响酶联免疫吸附试验+PSAM检测A？-42，t-tau的各种因素 和p-tau，并优化检测性能。开发一种通用的从 EL ISA至EL ISA+PSAM检测。 2.用最多3个参数验证优化EL ISA+PSAM方法的通用性 不同制造商为每种分析物提供的商业酶联免疫吸附试验试剂盒。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.