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。在我们的第二阶段项目中，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。特别是，我们将 1)设计并制作了PSAM预制照明装置；2)设计并研制了PSAM检测装置 系统试剂盒、EL ISA PSAM试剂盒用于检测A？-42、t-tau、p-tau、BDNF和proBDNF；3)进行广泛的 针对研究的绩效评估研究仅使用PSAM产品的应用。

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 II project, we propose to further develop a very sensitive and inexpensive immunoassay platform for detection of biomarkers for various neurodegenerative diseases. In particular, we will 1) design and manufacture a pre-production PSAM illumination device; 2) design and develop a PSAM detection system kit, ELISA + PSAM kits for detection of Aß-42, t-tau, p-tau, BDNF and proBDNF; 3) conduct extensive performance evaluation studies towards Research Use Only applications of the PSAM products.