Alzheimer's disease (AD), the most common form of dementia, is a complex disorder that is difficult to diagnose and treat. Currently, there is no readily available biomarker test panel to aid diagnosis or monitor disease progression. Although analysis of biomarkers in cerebrospinal fluid (CSF) has provided useful information, the validation of biomarkers in blood has not been as well-documented. The goal of this project is to develop a multiplex biomarker test for blood samples from Alzheimer's patients. In Phase I of this grant, we will demonstrate the feasibility of a novel rapid multiplex assay for serum/plasma biomarkers of AD. We have developed a proprietary method for using the real-time-polymerase chain reaction (PCR) technology to measure protein concentrations in a multiplexed fashion with very high sensitivity and reproducibility. The method, Multiplexed Real-Time-Immuno-PCR (MRI-PCR), consists of a sandwich immuno-assay in which the detection antibodies are labeled with different DNA sequences. Real-time PCR is then used to quantify all of the DNA labels in parallel, thus revealing the quantities of the cognate proteins in the biological samples. Preliminary data have demonstrated that multiplexed cytokine measurements employing MRI-PCR assays provides ~2 orders of magnitude more sensitive detection and >2 orders of magnitude increased dynamic range in comparison to standard ELISA assays, along with very low standard deviations (CV <10%) and high specificity. In Phase I studies, a panel of reagents to detect 15 human biomarkers of AD will be prepared and evaluated in the MRI-PCR assay. The target proteins will then be spiked into normal human serum to validate the assay in a physiological sample. Parameters to be evaluated include sensitivity, dynamic range, accuracy, precision, dose-response linearity, and specificity. A small pilot study will be conducted to evaluate the performance of the test on serum samples from AD patients as well as age-matched normal controls (n=10-20 samples). In Phase II, the assay will be further validated in a large scale clinical study to demonstrate the value of this technology in the diagnosis, prognosis and monitoring of therapy for AD. The results of these studies will lead to a new test for AD that is less invasive than CSF analysis and will provide valuable information to both clinicians as well as researchers.
This project will develop a novel assay to assist in the diagnosis, prognosis, and monitoring of therapy for Alzheimer's disease.
