Ultra-sensitive detection of prion protein fibrils by flow cytometry in blood from cattle affected with bovine spongiform encephalopathy

<p>Abstract</p> <p>Background</p> <p>The definite diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD) in humans or bovine spongiform encephalopathy (BSE) in cattle currently relies on the <it>post mortem </it>detection of the pathological form of the prion protein (PrP^Sc) in brain tissue. Infectivity studies indicate that PrP^Scmay also be present in body fluids, even at presymptomatic stages of the disease, albeit at concentrations well below the detection limits of currently available analytical methods.</p> <p>Results</p> <p>We developed a highly sensitive method for detecting prion protein aggregates that takes advantage of kinetic differences between seeded and unseeded polymerization of prion protein monomers. Detection of the aggregates was carried out by flow cytometry. In the presence of prion seeds, the association of labelled recombinant PrP monomers in plasma and serum proceeds much more efficiently than in the absence of seeds. In a diagnostic model system, synthetic PrP aggregates were detected down to a concentration of approximately 10^-8nM [0.24 fg/ml]. A specific signal was detected in six out of six available serum samples from BSE-positive cattle.</p> <p>Conclusion</p> <p>We have developed a method based on seed-dependent PrP fibril formation that shows promising results in differentiating a small number of BSE-positive serum samples from healthy controls. This method may provide the basis for an <it>ante mortem </it>diagnostic test for prion diseases.</p

Ultra-sensitive detection of prion protein fibrils by flow cytometry in blood from cattle affected with bovine spongiform encephalopathy-0

<p><b>Copyright information:</b></p><p>Taken from "Ultra-sensitive detection of prion protein fibrils by flow cytometry in blood from cattle affected with bovine spongiform encephalopathy"</p><p>BMC Biotechnology 2005;5():26-26.</p><p>Published online 4 Oct 2005</p><p>PMCID:PMC1266054.</p><p>Copyright © 2005 Trieschmann et al; licensee BioMed Central Ltd.</p>0 μl PBS (left panel) or in the same volume of serum (right panel), followed by flow cytometry. The measurements are depicted in a Fluorescence 1 (FL1-H) vs. Fluorescence 2 (FL2-H) dot-plot. The number of counts in the area containing specific signals (R2) is given in the figures. Aggregate formation in serum is strongly inhibited

Ultra-sensitive detection of prion protein fibrils by flow cytometry in blood from cattle affected with bovine spongiform encephalopathy-2

<p><b>Copyright information:</b></p><p>Taken from "Ultra-sensitive detection of prion protein fibrils by flow cytometry in blood from cattle affected with bovine spongiform encephalopathy"</p><p>BMC Biotechnology 2005;5():26-26.</p><p>Published online 4 Oct 2005</p><p>PMCID:PMC1266054.</p><p>Copyright © 2005 Trieschmann et al; licensee BioMed Central Ltd.</p>yzed by flow cytometry. The measurements are shown in a Fluorescence 1 (FL1-H) vs. Side-Scatter (SSC) dot-plot. All six BSE-samples (A-F) can be differentiated from the controls (G-J) by a population of events in region R3 (green dots). Panel K: Quantification of measurements shown in panels A-J

Ultra-sensitive detection of prion protein fibrils by flow cytometry in blood from cattle affected with bovine spongiform encephalopathy-1

<p><b>Copyright information:</b></p><p>Taken from "Ultra-sensitive detection of prion protein fibrils by flow cytometry in blood from cattle affected with bovine spongiform encephalopathy"</p><p>BMC Biotechnology 2005;5():26-26.</p><p>Published online 4 Oct 2005</p><p>PMCID:PMC1266054.</p><p>Copyright © 2005 Trieschmann et al; licensee BioMed Central Ltd.</p>(panel A) or presence (panel B) of 10nM PrP aggregates. Panel C: quantification of measurements shown in A and B, and of measurements (not shown) with different seed concentrations. The measurements are depicted in a Fluorescence 1 (FL1-H) vs. Side-Scatter (SSC) dot-plot. Aggregate formation (signal in region R1) was strongly enhanced by all seed concentrations tested, from 5 nM to 10nM