Highly sensitive immunoassay for determination of biomarkers for neurodegenerative diseases

用于测定神经退行性疾病生物标志物的高灵敏度免疫测定

基本信息

批准号：
10513251
负责人：
Simon Bystryak
金额：
$ 3.43万
依托单位：
ALLIED INNOVATIVE SYSTEMS, LLC
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10513251
关键词：
Achievement Affect Alzheimer&apos s Disease Alzheimer&apos s disease diagnosis Alzheimer’s disease biomarker Amyloid beta-42 Antibodies Award Bacteria Belief Biochemical Reaction Biological Assay Biological Markers Biology Blood Blood specimen Businesses Cerebrospinal Fluid Complex Coronavirus Detection Development Diagnosis Drug Screening Enzyme-Linked Immunosorbent Assay Enzymes European HIV Core Protein p24 Horseradish Peroxidase Immune System Diseases Immunoassay Interleukins Label Legal patent Licensing Lighting Malignant Neoplasms Manuals Manufacturer Name Measures Mediating Medicine Methods Monitor Neurodegenerative Disorders Neurofibrillary Tangles Performance Phase Phenylenediamines Plasma Process Property Proteins Public Health Reaction Reagent Reproducibility Sampling Secure Serum Signal Transduction Solid System Technology Testing Time Validation Virus Visible Radiation analytical method antibody detection base cost cost effective cytokine design detection assay detection limit detection method detection platform detection test early detection biomarkers improved innovation interest laboratory equipment new technology success tau Proteins tau-1

项目摘要

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.

抽象的该提案的长期目标是进一步开发光化学信号放大方法 (PSAM) 用于提高传统酶联免疫吸附测定 (ELISA) 的灵敏度测定各种神经退行性疾病的生物标志物，例如阿尔茨海默病 (AD)。 ELISA等免疫酶方法广泛应用于生物学和医学领域的药物筛选、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性疾病以及神经退行性疾病的生物标志物它们被常规使用。在执行大量测试的手动、半自动和自动化系统中。许多情况下 ELISA 的灵敏度不够。 AD 早期诊断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 ß-42 (Aß-42)，导致寡聚斑块、总 tau (t-tau) 和磷酸化 tau 蛋白的形成（p-tau）（它会增加细胞内神经原纤维缠结（NFT）。迄今为止，还没有敏感且成本高的方法定量这三种蛋白质的有效方法，阻碍了诊断和进展 AD 监测。现有的高灵敏度方法既麻烦又昂贵，因此越来越多。需要一个通用的 ELISA 平台来检测低水平的 Aß-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，不需要专门的治疗在我们的第一阶段项目中，我们建议进一步开发一种非常灵敏且廉价的实验室设备。用于检测各种神经退行性疾病生物标志物的免疫测定平台。 ELISA + PSAM 技术，包括两个步骤：（1）常规辣根过氧化物酶（HRP）介导测定 (ELISA)，其中使用常见的显色 HRP 底物，以及 (2) 含有酶促反应的产物受到可见光的照射，产生 DAP-。催化 DAP 浓度急剧增加（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 Aß-42、t-tau 检测性能的所有变量和 p-tau 并优化检测的性能，以开发一种通用的方法来进行转换。 ELISA 至 ELISA + PSAM 检测。 2. 通过使用多达 3 个来验证优化 ELISA + PSAM 检测的通用方法来自不同制造商的针对每种分析物的市售 ELISA 试剂盒这将包括测定。测定的灵敏度和再现性/精确度。