1a.Objectives (from AD-416):
The long term goal of this project is the development of rapid, reliable, and sensitive diagnostic assays for the identification of zoonotic viruses that are suitable for use in point-of-care facilities, as well as in the field for use during times of natural or intentionally caused disease epidemics.
1b.Approach (from AD-416):
The feasibility of this goal will be demonstrated in the proposed research by the development of Au nanoparticle-mediated indirect and direct capture assays for arboviruses including Rift Valley Fever Virus (RVFV) or antibodies to these viruses. The detection technology is based upon surface enhanced Raman scattering (SERS) spectroscopy, an analytical tool that is well known for its high detection specificity and sensitivity and which affords several advantages over other nucleic acid detection and immunological technologies currently in use. This includes the reduction in assay costs by affording sensitivity without current amplification technologies and easy adaptation to point-of-care use. The primary focus of the proposed studies will be the demonstration and evaluation of the relative efficacies of alternative in vitro model assays, followed by the translation of these findings to the development of an optimized assay for detecting genetic material, antigen or antibodies to arboviruses.
The objective of this project is to use nanotechnology to develop point of care (POC) diagnostic tools for insect transmitted diseases. In the past year, we have expanded upon our efforts to develop magnetic capture-based SERS immunoassays for the multiplex detection of West Nile virus (WNV) and Rift Valley Fever virus (RVFV) antigens. Assays were developed using Raman reporter-coated Au nanoparticles functionalized with polyclonal antibodies for the WNV envelope (E) protein and RVFV nucleocapsid N protein and paramagnetic nanoparticles functionalized with the same antibodies. Upon the addition of target antigen, GNP/antigen/PMP immuno-recognition complexes are formed which are then retrieved from solution by magnetic pull-down for 785 nm laser interrogation. 2-plex assays developed for simultaneous E and N detection provide a conservative limit of detection (LOD) of ~5 fg/ml and 3-plex assays which include nanoparticle reagents developed for the detection of the F1 antigen of Yersinia pestis provide similar LODs. These assays significantly exceed the LODs typically recorded for ELISAs and provide an assay format that can be easily adapted for field or POC testing. Currently, we are characterizing immunological reagents for the VP7 antigens of both the Epizootic Hemmorhagic Disease and Bluetongue viruses and will develop and optimize a 4-plex immunoassay that is specifically intended for the sensitive and discriminate detection of zoonotic viruses in biological specimens.
As part of our effort to adapt these assays to realistic test conditions, we have also begun to develop and optimize assays that are conducted using simulants of biological test specimens. Although we have found that assays containing high concentrations of certain simulants (e.g. fetal bovine serum) demonstrate reductions in LOD sensitivity, antigen discrimination is unaffected and dilution of the test sample mitigates reductions in detection sensitivity. We have also made significant progress in engineering a handheld Raman spectrometer for magnetic capture capabilities. In these studies, magnets are positioned within a vial holder attachment such that magnetically concentrated immuno-recognition complexes are optimally aligned for laser interrogation. According to our currently envisioned assay platform, biological test samples will be added to lyophilized NP reagents, which will the be briefly incubated with mild agitation and then placed into the magnetic vial holder attachment for laser excitation and data acquisition.