Detection of misfolded prion protein in blood with conformationally sensitive peptides.
BACKGROUND
The long-standing goal of a preclinical diagnostic test for transmissible spongiform encephalopathy (TSE) has recently become urgent because of the discovery that humans with variant Creutzfeldt-Jakob disease can transmit disease via blood transfusions.


METHODS
The misfolded protein diagnostic (MPD) assay employs a pyrene-labeled palindromic sequence of prion peptides that undergoes a cascade of coil to beta-sheet conversion in the presence of the misfolded prion protein (PrP(TSE)). The ability of the assay to detect PrP(TSE) in brain, serum, and plasma was tested. The basic protocol involved a several-hour incubation of 200-microL sample volumes with the peptide reagent in 96-well plates, after which fluorescence was monitored by a fluorescence plate reader with an excitation wavelength of 350 nm and emission scanning wavelength range of 365 to 600 nm.


RESULTS
Target specificity for PrP(TSE) was documented by correlation of assay signal with Western blot signals in brain tissue from TSE-infected, normal, and knockout mice and negative assay signals by use of reagents with different peptide sequences. When applied to plasma or serum, the assay discriminated between samples from a variety of experimental and natural TSE infections compared to uninfected controls, with a sensitivity threshold of approximately 1 infectious dose per mL in pooled plasma from TSE-infected mice.


CONCLUSIONS
The MPD assay is a sensitive and specific test for the detection of PrP(TSE) that may be useful in both preclinical and clinical diagnosis of TSE diseases of animals and humans.
Pan
Tao
T
Adlyfe Inc., 9430 Key West Avenue, Rockville, MD 20850, USA.
Sethi
Jasmeet
J
Nelsen
Craig
C
Rudolph
Alan
A
Cervenakova
Larisa
L
Brown
Paul
P
Orser
Cindy S
CS
D000595:Amino Acid Sequence; D000818:Animals; D007562:Creutzfeldt-Jakob Syndrome; D004195:Disease Models, Animal; D005453:Fluorescence; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011328:Prions / Q000097:blood* / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D012453:Saimiri; D012680:Sensitivity and Specificity; D012756:Sheep
D011328:Prions