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Protein Misfolding Cyclic Amplification of Disease‐Associated Prion Protein Using Human Autopsy Tissues
Author(s) -
Jones M.,
Peden A.,
Ironside J.,
Prowse C.,
MacGregor I.,
Head M.
Publication year - 2006
Publication title -
transfusion medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.471
H-Index - 59
eISSN - 1365-3148
pISSN - 0958-7578
DOI - 10.1111/j.1365-3148.2006.00693_13.x
Subject(s) - scrapie , blot , prion protein , hamster , human brain , in vitro , microbiology and biotechnology , western blot , biology , immunoassay , antibody , virology , pathology , medicine , disease , biochemistry , immunology , gene , neuroscience
The protein misfolding cyclic amplification (PMCA) technique allows for the in vitro production of protease‐resistant prion protein (PrP res ) using a seed of disease‐associated prion protein (PrP sc ) to convert normal cellular prion protein (PrP c ). This system has been developed using brain tissue from scrapie infected hamsters to provide the seed and normal uninfected hamster brain to provide PrP c substrate and the required conversion factors. These are mixed and subjected to repeated rounds of sonication and incubation. Extremely low concentrations of PrP sc , such as those present in blood, have been detectable. In order to adapt this method for use in CJD research and in the development of diagnostic tests for human prion diseases it will be necessary to identify a source of human PrP c and co‐factors that support amplification of human PrP sc . We have tested whether human autopsy tissues held at the National CJD Surveillance Unit support PMCA. Using a seed extract from variant CJD brain mixed with homogenates of nonCJD neurological control brain we have been able to effect a >600‐fold increase in the detection limit of PrP res , as determined by Western Blotting. Further development of this technique in concert with high sensitivity PrP sc detection methods, such as conformation dependent immunoassay, may result in a method capable of acting as a confirmatory assay for disease‐associated prion protein in human tissues and biological fluids such as blood.