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From: TSS ()
Subject: Detection of misfolded prion protein in blood with conformationally sensitive peptides
Date: July 29, 2007 at 10:02 am PST

TRANSFUSION COMPLICATIONS

Detection of misfolded prion protein in blood with conformationally sensitive peptides

Tao Pan , Jasmeet Sethi , Craig Nelsen , Alan Rudolph , Larisa Cervenakova , Paul Brown , and Cindy S. Orser From Adlyfe Inc., Rockville; the American Red Cross Holland Laboratory, Rockville; and 7815 Exeter Road, Bethesda, Maryland
Dr Cindy Orser, Adlyfe Inc., 9430 Key West Avenue, Rockville, MD 20850; e-mail: corser@adlyfe.com.
This work was partially supported by DARPA DSO Contract N66001-02-C-8001 and NHLBI Grant 1R44 HL070399-03 to CSO.

Abstract

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.

STUDY DESIGN AND METHODS: The misfolded protein diagnostic (MPD) assay employs a pyrene-labeled palindromic sequence of prion peptides that undergoes a cascade of coil to ‚-sheet conversion in the presence of the misfolded prion protein (PrPTSE). The ability of the assay to detect PrPTSE in brain, serum, and plasma was tested. The basic protocol involved a several-hour incubation of 200-ĶL 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 PrPTSE 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 PrPTSE that may be useful in both preclinical and clinical diagnosis of TSE diseases of animals and humans.


http://www.blackwell-synergy.com/doi/abs/10.1111/j.1537-2995.2007.01284.x

TSS



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