Discrimination between prion-infected and normal blood samples by protein misfolding cyclic amplification.
BACKGROUND
Diagnosis of prion disease from blood samples requires the detection of minute quantities of misfolded protein (PrP(Sc)) against a high background of correctly folded material (PrP(C)). Protein misfolding cyclic amplification (PMCA) is a technique that can amplify small amounts of seed PrP(Sc) to a level detectable by conventional methods. Application of PMCA to the testing of whole blood samples enhances the ability to detect PrP(Sc) and allows antemortem detection of prion infection and could facilitate blood screening.


METHODS
The PMCA method was used to detect prion infection in blood samples obtained from mice experimentally infected with prion disease. Mice were culled at various time points throughout the incubation period for disease and subjected to serial PMCA (sPMCA). Amplified samples were then analyzed by Western blotting to confirm the presence or absence of infection.


RESULTS
After sPMCA, blood samples from Rocky Mountain Laboratory-infected mice showed amplification of PrP(Sc) to levels readily detectable by Western blotting. Control samples obtained from mice mock inoculated with sterile phosphate-buffered saline did not yield any amplification products.


CONCLUSIONS
sPMCA performed on small volumes of whole blood gave amplification of PK-resistant material to a level detectable by standard methods. Discrimination between infected and control samples was achieved without the need for processing or fractionation of whole blood. The use of whole blood as an analyte circumvents the need to identify the optimal blood compartment for analysis and guarantees the totality of misfolded PrP will be available for detection.
Tattum
M Howard
MH
MRC Prion Unit and Department of Neurodegenerative Disease, Institute of Neurology, University College London, London, UK.
Jones
Samantha
S
Pal
Suvankar
S
Collinge
John
J
Jackson
Graham S
GS
D000818:Animals; D007562:Creutzfeldt-Jakob Syndrome / Q000097:blood / Q000175:diagnosis*; D019286:Endopeptidase K; D006801:Humans; D051379:Mice; D018620:PrPSc Proteins / Q000097:blood* / Q000737:chemistry; D017510:Protein Folding
D018620:PrPSc Proteins; D019286:Endopeptidase K