|Neng, J - University Of Wyoming|
|Harpster, Mark - University Of Wyoming|
|Zhang, H - University Of Wyoming|
|Mecham, James - US Department Of Agriculture (USDA)|
|Wilson, William - Agricultural Marketing Service (AMS, USDA)|
|Johnson, P - University Of Wyoming|
Submitted to: Biosensors and Bioelectronics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/12/2010
Publication Date: 11/15/2010
Citation: Neng, J., Harpster, M.H., Zhang, H., Mecham, J.O., Wilson, W.C., Johnson, P.A. 2010. A versatile SERS-based immunoassay for immunoglobulin detection using antigen-coated gold nanoparticles and malachite green-conjugated protein A/G. Biosensors and Bioelectronics. 26(3):1009-15. DOI: 10.1016/j.bios.2010.08.015.
Interpretive Summary: A novel nanopartical system was developed for detection of specific antibodies in serum. The system utilizes gold nanoparticles coated with a West Nile virus protein and a specific type of dye bound to a protein A/G molecule that interacts with antibodies. When the specific antibody is present in a solution with the gold nanoparticles and the dye-bound protein A//G then a signal can be detected using a relatively inexpensive instrument that detects the surface enhanced Raman scattering (SERS). The sensitivity and inherent versatility of the assay, which is provided by the binding of pA/G to a broad spectrum of immunoglobulins in different mammalian species, suggests that it could be developed as an alternative immunoassay format to the ELISA.
Technical Abstract: A surface enhanced Raman scattering (SERS) immunoassay for antibody detection in serum is described in the present work. The developed assay is conducted in solution and utilizes Au nanoparticles coated with the envelope (E) protein of West Nile Virus (WNV) as the SERS-active substrate and malachite green (MG)-conjugated protein A/G (MG-pA/G) as a bi-functional Raman tag/antibody binding reporter. Upon incubation of these reagents with serum collected from rabbits inoculated with E antigen, laser interrogation of the sandwiched immunocomplex revealed a SERS signaling response diagnostic for MG. The intensification of signature spectral peaks is shown to be proportionate to the concentration of added serum and the limit of antibody detection is 2 ng/ml of serum. To assess assay performance relative to more a traditional immunoassay, indirect enzyme-linked immunosorbent assays conducted using the same concentrations of reagents were found to be >400-fold less sensitive. Quartz crystal microbalance with dissipation (QCM-D) monitoring of immunocomplex film deposition on solid Au surfaces also confirmed the formation of antigen-antibody-protein A/G trilayers and provided quantitative measurements of film thickness which likely position MG within the sensing distance of laser-elicited, enhanced electromagnetic fields. The sensitivity and inherent versatility of the assay, which is provided by the binding of pA/G to a broad spectrum of immunoglobulins in different mammalian species, suggests that it could be developed as an alternative immunoassay format to the ELISA.