Capillary electrophoresis-based noncompetitive immunoassay for the prion protein using fluorescein-labeled protein A as a fluorescent probe.
A novel CE-based noncompetitive immunoassay for prion protein (PrP) was established. Fluorescein isothiocyanate (FITC)-labeled protein A (FITC-PrA) was used as a fluorescent probe to tag monoclonal antibody through noncovalent binding of FITC-PrA to the Fc region of the antibody. The FITC-PrA-Ab was incubated with the analyte, prion protein, under optimized condition, forming the immunocomplex FITC-PrA-Ab-PrP. The complex was separated and analyzed by capillary zone electrophoresis. The addition of carboxymethyl-beta-cyclodextrin in the running buffer as dynamical coating reagent improved the reproducibility and the resolution. The complex was isolated in less than 1 min with theoretical plates of 3.8 x 10(4). Relative standard deviations of peak height and migration time for the complex were 3.46 and 1.48%, respectively. A linear relationship was established for the bovine recombinant prion protein (rPrP) concentration in the range from 0.2 to 2.0 mug/mL and the peak height. The correlation factor was r2 = 0.9969. The estimated detection limit for rPrP was approximately 6 ng/mL, which is 3 times the signal-to-noise ratio. The method was successfully applied for testing blood samples from scrapie-infected sheep.
Yang
Wen-Chu
WC
Department of Chemistry, Iowa State University, Ames, Iowa 50011, Ames Laboratory USDOE, Iowa State University, Ames, Iowa 50011, USA.
Schmerr
Mary Jo
MJ
Jackman
Roy
R
Bodemer
Walter
W
Yeung
Edward S
ES
D000345:Affinity Labels; D000818:Animals; D000915:Antibody Affinity; D001666:Binding Sites, Antibody; D019075:Electrophoresis, Capillary / Q000379:methods*; D019793:Fluorescein / Q000737:chemistry*; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D011328:Prions / Q000737:chemistry*; D011994:Recombinant Proteins; D012756:Sheep / Q000097:blood; D013205:Staphylococcal Protein A / Q000737:chemistry*
D000345:Affinity Labels; D011328:Prions; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D019793:Fluorescein