Submitted to: Analytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/29/2005
Publication Date: 10/1/2005
Citation: Driskell, J.D., Kwarta, K.M., Lipert, R.J., Porter, M.D., Neill, J.D., Ridpath, J.F. 2005. Low level detection of viral pathogens by a surface-enhanced Raman scattering based immunoassay. Analytical Chemistry. 77(19):6147-6154. Interpretive Summary: Historically, diagnostic tests for viruses have been based on the binding of antibodies produced against viral proteins to viruses contained in sample material. Many highly specific antibodies have been generated for use in the detection of viruses. The traditional methods used to detect the binding of antibodies to viral proteins, known as readout technologies, are cumbersome, require extensive sample preparation and do not allow for the use of multiple antibodies against different viruses in the same test. This study explores the use of a new readout technology called surface-enhanced Raman scattering (SERS). The model system used to develop this technology was the binding of antibodies to feline calicivirus. It was found that SERS works well as a readout technology for detecting the presence of a virus. The advantage of this technology is that it eliminates the need for extensive sample preparation. This study represents the initial phase of this research. This research will be extended in the future to address increased sensitivity, reducing time needed for tests and using multiple antibodies in one test. These studies indicate that tests based on SERS detection would be adaptable for use in rapidly differentiating viruses in the event of disease outbreaks.
Technical Abstract: The need for rapid, highly sensitive, and versatile diagnostic tests for viral pathogens spans from human and veterinary medicine to bioterrorism prevention. As an approach to meet these demands, a diagnostic test employing monoclonal antibodies (mAbs) for the selective extraction of viral pathogens from a sample in a sandwich immunoassay format has been developed using surface-enhanced Raman scattering (SERS) as a readout method. The strengths of SERS-based detection include its inherent high sensitivity and facility for multiplexing. The capability of this approach is demonstrated by the capture of feline calicivirus (FCV) from cell culture media that is exposed to a gold substrate modified with a covalently immobilized layer of anti-FCV monoclonal antibodies. The surface-bound FCVs are subsequently coupled with an extrinsic Raman label (ERL) for identification and quantification. These ERLs consist of 60-nm gold nanoparticles coated first with a layer of Raman reporter molecules and then a layer of mAbs. The Raman reporter molecule is strategically designed to chemisorb as a thiolate adlayer on the gold nanoparticle, to provide a strong and unique spectral signature, and to covalently link a layer of mAbs to the gold nanoparticle. The last feature provides a means to impart the bioselectivity necessary to tag substrate-bound FCV. This paper describes the development of this chip-scale assay, which uses cell culture media as a sample matrix, has a linear dynamic range of 1 x 10**6 to 2.5 x 10**8 viruses/mL, and a limit of detection of 1 x 10**6 viruses/mL. These results reflect the findings of a detailed series of investigations on the effects of salt concentration, ERL binding buffer, blocking buffer, surfactant, and sample agitation, all of which were aimed at minimizing nonspecific binding, maximizing FCV binding efficiency, and improving the detection limit.