NR ATKJ
AU Yang,W.C.; Yeung,E.S.; Seemann,S.K.; Bodemer,W.; Schmerr,M.J.
TI A Fluorescence Immunoassay for the Prion Protein Using Fluorescein Labeled Protein A
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Diagnosis DIA-21
PT Konferenz-Poster
AB Fluorescence immunoassays (FIA) have been used to detect the endogenous prion protein in the blood of TSE infected sheep. These assays were analyzed by capillary electrophoresis using laser-induced fluorescence detection (CE-LIF) and were competitive assays using fluorescent peptides from the prion protein and corresponding antibodies. Both monoclonal and polyclonal antibodies have been used. To further exploit the advantages of FIA for detection of the prion protein, we investigated a noncompetitive FIA for prion protein by using the ability of protein A to bind to the Fc region of mammalian immunoglobulins. Initially, a well characterized monoclonal antibody, 12F10, was incubated with fluorescein labeled protein A, and bovine recombinant prion protein (rPrP) in 20mM Tricine containing 0.1% BSA, pH 8.0 at 25C for 30 min and then analyzed by CE-LIF using the running buffer 20 mM TAPS containing 0.5% CM-beta-CD at pH 8.8. When 27kV was used with a 50 micrometer(I.D.) x 30 cm fused silica capillary, a well-defined immunocomplex peak with a number of theoretical plates equal to 25,000 at the migration time of approximately 0.9 min was observed. The system was reproducible with relative standard deviations of peak height and migration time for the complex 1.48% and 3.46%, respectively. A linear relationship with a correlation factor of 0.9969 was established between rPrP concentration and the peak height. The method was used to test the blood samples from normal and scrapie-infected sheep, and the results correlated with those obtained from Western blot analysis of brain samples from sheep taken post-mortem to establish their scrapie status. Other monoclonal antibodies were evaluated for their ability to function in this free solution assay and have worked in the same fashion as 12F10. This approach has added another method that is rapid and robust to detect the endogenous prion protein in blood of scrapie infected sheep and potentially other species.
AD Wen Chu Yang, Edward S. Yeung, Sarah K. Seemann, Mary Jo Schmerr, Department of Chemistry, Iowa State University, Ames, IA, USA; Walter Bodemer, German Primate Center, Göttingen, D37077, Germany
SP englisch
PO Deutschland