Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801
Performing Department
Veterinary Research & Extension
Non Technical Summary
The goal of this lab's contribution to Multistate Project NC1180 "Control of Emerging and Re-emerging Poultry Respiratory Diseases in the United States" is to collaborate on the development of a surface-enhance Raman spectroscopy (SERS) based avian influenza virus diagnostic assay that will allow for rapid detection of avian influenza and allow the capability for multiplexing for detection of multiple influenza virus hemagluttinin subtypes simultaneously. This project fits with the second objective of NC1180 multistate project which is to "develop improved diagnostic capabilities including real time PCR as well as other rapid on-farm tests for economically important respiratory diseases". A diagnostic method for rapid, in the field subtyping of outbreaks of avian influenza virus in poultry is a critical area in need of development that would aid in rapid response and risk assessment of an avian influenza outbreak in poultry. Current techniques including PCR and microarrays are sensitive and specific, but theses assays lack the capacity for field deployment for rapid results and limited true multiplexing capability. A SERS-based immunoassay platform requires the capture of an antigen on an antibody-modified gold substrate (gold-plated capture filter) in the first step, and selective tagging of the captured antigen with functionalized gold nanoparticles in the second step. Central to this technology are the extrinsic Raman labels (ERLs) used to tag surface-immobilized antigen for quantification. Each ERL consists of a gold nanoparticle modified with an antibody and a Raman reporter molecule. SERS provides many advantages compared to traditional enzymatic or fluorescence based detection. SERS is not prone to photobleaching, enables simultaneous multiplexed detection, and can achieve single molecule detection. The collaborative lab (J. Driskell) that will be performing these SERS assays have successfully applied SERS for the detection of proteins and viruses. Furthermore, studies in this laboratory have demonstrated the ability to detect a single ERL, showcasing the potential of this technique to detect a single pathogen. This lab has recently used rotation-induced flux for the antigen labeling step as well as the antigen capture step and has reduced the incubation time of this assay from 24 hours to 25 minutes, demonstrating a technique that rapidly reduces the time required for the assay. The technology to miniaturize Raman instrumentation for portable and on-site analysis exists and several commercial instrumentation manufacturers now offer handheld devices.
Animal Health Component
40%
Research Effort Categories
Basic
20%
Applied
40%
Developmental
40%
Goals / Objectives
Develop improved diagnostic capabilities including real time PCR as well as other rapid on-farm tests for economically important respiratory diseases.
Investigate the pathogenesis and polymicrobial interactions of specific infectious agents associated with poultry respiratory diseases (this includes interactions with underlying immunosuppressive agents).
Project Methods
Development of a SERS based multiplexed avian influenza virus assay for poultry requires stocks of multiple subtypes of avian influenza virus and methods to titer the viruses, primarily 50% egg infectious dose (EID50). Multiple different low pathogenic avian influenza virus isolates that are different subtypes (H1-H13, those that have the capacity to infect poultry) will be propagated in 9-day-old embryonated chicken eggs and also titered using hemagluttination assay and EID50 after propagation for viral stocks to be used in the subsequent experiments. These viruses will be used for testing the SERS platform. The PI (E. Driskell) has a strong background in avian influenza virus research and the appropriate techniques (avian influenza virus isolation, propagation, and quantification) are established in our lab at University of Illinois.Given that this work proposes to capture intact virus, it is necessary to screen available antibodies for binding to the intact viral target. We will use an ELISA, an established technique, to screen antibodies and select only those that bind poultry influenza virus isolates of interest for further investigation with the collaborative lab's gold-plated capture filters. For this assay, Immulon 2HB plates will be coated with whole virus, blocked, and then serial dilutions of the primary antibody being screened will be applied to the plate. Appropriate secondary antibody (HRP labeled goat anti-mouse IgG H & L chains) will be applied and then the reaction will be stopped with sulfuric acid and absorbance of plates read on a SpectraMax Plus at 450 nm emission. Our lab (E. Driskell) has already successfully performed ELISA on H1 subtype influenza viruses A/PR/8/34 and A/New Caledonia/20/99 to evaluate binding of a panel of different anti-H1 antibodies.The SERS platform will be tested for the ability to detect different hemagluttinin (HA) subtypes of influenza virus. In the first stages of the study, several different antibodies specific for two different HA subtypes of interest will be screened via ELISA. These antibodies will be screened against two different poultry isolates of the same HA subtype to ensure the antibody binds the same subtype, regardless of isolate. Additionally, these antibodies will be screened against different HA influenza virus subtypes to ensure there is no binding to different HA subtypes, indicating that the antibody chosen is truly subtype specific.Once the appropriate antibodies are selected, the collaborative laboratory (J. Driskell) will apply them in the SERS platform, using the antibody-modified gold substrate, the poultry influenza virus of interest, and ERLs for viral detection. After the SERS platform is confirmed to work independently for two different HA subtypes of influenza virus, multiplexing of two HA subtypes will be explored by simultaneous use of two different antibodies.