Highly sensitive immunoassay for determination of biomarkers for neurodegenerative diseases

摘要 这项建议的长远目标是进一步发展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（AAP14 - 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的生物标志物的免疫测定平台。特别是要 1)设计和制造了一个预生产的PSAM照明装置; 2）设计和开发了一个PSAM检测 系统试剂盒，ELISA + PSAM试剂盒用于检测Ablu-42、t-tau、p-tau、BDNF和proBDNF; 3）进行广泛的 PSAM产品仅供研究使用的性能评估研究。

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.