Development of a nanoparticle-based surface-modified fluorescence assay for the detection of prion proteins.
A nanoparticle-based immunoassay for the detection of recombinant bovine prion protein (PrP) was developed as a step in the development of screening tools for the prevention of the spread of transmissible spongiform encephalopathies. The assay is based on the competitive binding between PrP and a peptide-fluorophore to a nanoparticle-labeled antibody which is specific for a conserved prion sequence. The fluorophore, when bound to the antibody, is subject to surfaced-modified fluorescence, enabling detection of changes in the concentration of bound fluorophore in the presence of prion protein. Important factors considered during the development of the assay were ease of use, robustness, and detection level. The effects of pH and nanoparticle conjugation chemistry on surface-modified fluorescence observed in the assay were explored. Effects of concentrations of antibody and fluorophore on reproducibility and detection limits were examined. At present, the detection limits of the system are approximately equal to the antibody-peptide fluorophore equilibrium dissociation constant, which is near one nanomolar concentration. Improved assay performance could be obtained by optimization of the nanoparticle surface resonance effects. The simplicity of the assay and ease of use may make the type of assay described in this report attractive for screening purposes in the food industry.
Henry
James
J
Department of Chemical Engineering, Texas A&M University, College Station, TX 77843, USA.
Anand
Ashish
A
Chowdhury
Mustafa
M
Coté
Gerard
G
Moreira
Rosana
R
Good
Theresa
T
D005240:Feasibility Studies; D005456:Fluorescent Dyes; D005470:Fluorometry; D006046:Gold / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007118:Immunoassay / Q000379:methods*; D036103:Nanotechnology / Q000379:methods; D010316:Particle Size; D011328:Prions / Q000032:analysis* / Q000276:immunology; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology; D013499:Surface Properties
D005456:Fluorescent Dyes; D011328:Prions; D011994:Recombinant Proteins; D006046:Gold