|Title||Human platelets as a substrate source for the in vitro amplification of the abnormal prion protein (PrP) associated with variant Creutzfeldt-Jakob disease.|
|Publication Type||Journal Article|
|Year of Publication||2009|
|Authors||Jones M, Peden AH, Yull H, Wight D, Bishop MT, Prowse CV, Turner ML, Ironside JW, MacGregor IR, Head MW|
|Date Published||2009 Feb|
|Keywords||Blood Platelets, Brain Chemistry, Codon, Creutzfeldt-Jakob Syndrome, Humans, Immunoassay, Nucleic Acid Amplification Techniques, Polymorphism, Genetic, Protein Conformation, Protein Folding, PrPSc Proteins, Sensitivity and Specificity, Substrate Specificity|
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.
STUDY DESIGN AND 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.
|Grant List||G0600953 / / Medical Research Council / United Kingdom |
G0900580 / / Medical Research Council / United Kingdom