Human platelets as a substrate source for the in vitro amplification of the abnormal prion protein (PrP) associated with variant Creutzfeldt-Jakob disease.
BACKGROUND
Four recent cases of transfusion-related transmission of variant Creutzfeldt-Jakob disease (vCJD) highlight the need to develop a highly sensitive and specific screening test to detect infectivity in the blood of asymptomatic infected individuals. Protein misfolding cyclic amplification (PMCA), a method for the amplification of minute amounts of disease-associated abnormal prion protein (PrP(Sc)) to readily detectable levels, could be incorporated into such a test provided that a suitable substrate source for routine use in human PMCA reactions can be found.


METHODS
With the use of seed sources from individuals with variant and sporadic CJD, the use of human platelets (PLTs) as a PMCA substrate source was evaluated. The effects of seed/substrate prion protein gene (PRNP) codon 129 genotype compatibility on amplification efficiency and freeze-thaw on a substrate's ability to support amplification and the degree of amplification achieved by serial PMCA (sPMCA) were investigated.


RESULTS
Seed/substrate PRNP codon 129 compatibility was found to have a major influence on PrP(Sc) amplification efficiency. Individual substrates, of the same PRNP codon 129 genotype, could be pooled and stored frozen for use in subsequent PMCA reactions. A consistent 10-fold increase in PrP(Sc) detection sensitivity was achieved after each round of sPMCA, resulting in a 10,000-fold increase in detection sensitivity after four rounds, with no evidence of de novo PrP(Sc) production detected in the unseeded PLT substrate.


CONCLUSIONS
Providing issues of seed/substrate PRNP codon 129 compatibility are taken into consideration human PLTs are a suitable, readily available, renewable substrate source for use in human PMCA applications.
Jones
Michael
M
University of Edinburgh, Western General Hospital, UK. mjones1@staffmail.ed.ac.uk
Peden
Alexander H
AH
Yull
Helen
H
Wight
Darren
D
Bishop
Matthew T
MT
Prowse
Chris V
CV
Turner
Marc L
ML
Ironside
James W
JW
MacGregor
Ian R
IR
Head
Mark W
MW
D001792:Blood Platelets / Q000378:metabolism*; D001923:Brain Chemistry; D003062:Codon; D007562:Creutzfeldt-Jakob Syndrome / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007118:Immunoassay / Q000379:methods; D021141:Nucleic Acid Amplification Techniques; D011110:Polymorphism, Genetic; D018620:PrPSc Proteins / Q000032:analysis / Q000235:genetics*; D011487:Protein Conformation; D017510:Protein Folding; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D003062:Codon; D018620:PrPSc Proteins