Highly sensitive immunoassay for determination of biomarkers for neurodegenerative diseases

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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.