title: A versatile SERS-based immunoassay for immunoglobulin detection using antigen-coated gold nanoparticles and malachite green-conjugated protein A/G.
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, suggest that it could be developed as an alternative immunoassay format to the ELISA.
authors.affiliation: Molecular and Cellular Life Sciences Program, University of Wyoming, Laramie, WY 82071, United States.
authors.forename: Jing
authors.initials: J
authors.lastname: Neng
authors.forename: Mark H
authors.initials: MH
authors.lastname: Harpster
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.forename: James O
authors.initials: JO
authors.lastname: Mecham
authors.forename: William C
authors.initials: WC
authors.lastname: Wilson
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Johnson
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis; D000956:Antigens, Viral; D001426:Bacterial Proteins; D015374:Biosensing Techniques / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D017979:Gold Colloid; D007118:Immunoassay / Q000379:methods*; D007136:Immunoglobulins / Q000032:analysis*; D053768:Metal Nanoparticles; D058525:Quartz Crystal Microbalance Techniques; D011817:Rabbits; D012394:Rosaniline Dyes; D013059:Spectrum Analysis, Raman; D014902:West Nile virus / Q000276:immunology
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D001426:Bacterial Proteins; D017979:Gold Colloid; D007136:Immunoglobulins; D012394:Rosaniline Dyes; C070001:protein AG-gold complex; C005095:malachite green