Rapid, highly sensitive and specific, flexible, simple and cost-effective diagnostic techniques to detect and differentiate the causes of infections are critically needed to distinguish dangerous infections from common ones. We propose developing a simple multiplexed assay to identify the causes of viral fevers. Technologies that derive from recent advances in nanoscale engineering and micro-fluidics present synergies that can be leveraged in the development of multiplexed assays in large-scale screening applications. A simple and robust platform for quantitative multiprotein immunoanalysis has been developed with the use of magnetic core/shell nanoparticles (MLNPs) as a carrier. The magnetic properties of the MLNPs allow their manipulation by an external magnetic field in the separation and washing steps in the immunoassay;enhanced binding kinetics are also provided by the oscillating magnetic field. The luminescent properties arise from the the addition of lanthanide elements to oxide hosts or the addition of lanthanide chelates to porous silica hosts. Silver nano-islands within the latter particles provide significant enhancement of signal leading to the possibility of single particle detection. The optical emission from the particles also provides an internal calibration of the detection system this is important in reducing uncertainties in particle-based assays. Multiplexed sandwich immunoassay involves dual binding events on the surface of the MLNPs functionalized with the capture antibodies. Secondary antibodies labeled with conventional organic dyes (Alexa Fluor) can be used as reporters. Alternatively, the secondary label can be very small porous silica particles with chelate emission enhanced with silver, for a much greater signal and hence sensitivity. The amount of the bound secondary antibody is directly proportional to the concentration of the analyte in the sample. In our approach, the fluorescence intensity of the reporter dye is related to the luminescence signal of the MLNPs. In this way, the intrinsic luminescence of the MLNPs serves as an internal standard in the quantitative immunoassay. This technology will be implemented in a micro-fluidic system that will be automated. We will utilize reagents developed for Rift Valley fever virus (RVFV) detection to develop a prototype assay that will detect RVFV antibody and antigen. We will also develop monoclonal antibodies and recombinant proteins to three other viruses that cause hemorrhagic fever in humans: Dengue, Lassa, and Ebola virus. Using these reagents, we will develop a prototype test that will identify samples from individuals infected with these viruses. Validation with experimental and clinical samples will begin in the final year of the grant.
|Effective start/end date||8/16/10 → 7/31/12|
- National Institutes of Health: $892,251.00
- Immunology and Microbiology(all)