In vitro amplification and detection of variant Creutzfeldt-Jakob disease PrPSc.
Variant Creutzfeldt-Jakob disease (vCJD) poses a serious risk of secondary transmission and the need to detect infectivity in asymptomatic individuals is therefore of major importance. Following infection, it is assumed that minute amounts of disease-associated prion protein (PrP(Sc)) replicate by conversion of the host cellular prion protein (PrP(C)). Therefore, methods of rapidly reproducing this conversion process in vitro would be valuable tools in the development of such tests. We show that one such technique, protein misfolding cyclic amplification (PMCA), can amplify vCJD PrP(Sc) from human brain tissue, and that the degree of amplification is dependent upon the substrate PRNP codon 129 polymorphism. Both human platelets and transgenic mouse brain are shown to be suitable alternative substrate sources, and amplified PrP(Sc) can be detected using a conformation-dependent immunoassay (CDI), allowing the detection of putative proteinase K sensitive forms of PrP(Sc).
Jones
M
M
National CJD Surveillance Unit, School of Molecular and Clinical Medicine (Pathology), University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK. mjones@staffmail.ed.ac.uk
Peden
A H
AH
Prowse
C V
CV
Gröner
A
A
Manson
J C
JC
Turner
M L
ML
Ironside
J W
JW
MacGregor
I R
IR
Head
M W
MW
D000818:Animals; D001792:Blood Platelets; D015153:Blotting, Western / Q000379:methods; D001923:Brain Chemistry; D003062:Codon; D007562:Creutzfeldt-Jakob Syndrome / Q000175:diagnosis* / Q000378:metabolism; D005838:Genotype; D006801:Humans; D007118:Immunoassay / Q000379:methods; D051379:Mice; D008822:Mice, Transgenic; D021141:Nucleic Acid Amplification Techniques; D011110:Polymorphism, Genetic; D018620:PrPSc Proteins / Q000032:analysis / Q000235:genetics*; D011487:Protein Conformation; D017510:Protein Folding
D003062:Codon; D018620:PrPSc Proteins