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-β-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 × 104. 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 μg/mL and the peak height. The correlation factor was r 2 = 0.9969. The estimated detection limit for rPrP was ∼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.