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Published on: September 29, 2017
In vitro amplification and detection of variant Creutzfeldt-Jakob disease PrPSc
M Jones1, A H Peden, C V Prowse
1National 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
Abstract:
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).
Insights
Detecting variant Creutzfeldt-Jakob disease (vCJD) in asymptomatic individuals is crucial. Protein misfolding cyclic amplification (PMCA) shows promise for amplifying vCJD prions, aiding in early detection test development.
Area of Science:
- Neuroscience
- Infectious Diseases
- Biochemistry
Background:
- Variant Creutzfeldt-Jakob disease (vCJD) presents a significant risk for secondary transmission.
- Detecting prion infectivity in asymptomatic individuals is critical for public health.
- Prion diseases involve the misfolding and replication of prion proteins.
Purpose of the Study:
- To evaluate the efficacy of protein misfolding cyclic amplification (PMCA) for amplifying disease-associated prion protein (PrP(Sc)) from vCJD.
- To assess the influence of PRNP codon 129 polymorphism on PrP(Sc) amplification.
- To identify alternative substrate sources for PrP(Sc) amplification and detection.
Main Methods:
- Protein misfolding cyclic amplification (PMCA) technique was employed.
- Human brain tissue, platelets, and transgenic mouse brain were used as substrates.
- Conformation-dependent immunoassay (CDI) was utilized for PrP(Sc) detection.
Main Results:
- PMCA successfully amplified vCJD PrP(Sc) from human brain tissue.
- The extent of amplification was influenced by the PRNP codon 129 polymorphism.
- Human platelets and transgenic mouse brain proved to be effective alternative substrates.
- CDI enabled detection of proteinase K-sensitive forms of PrP(Sc).
Conclusions:
- PMCA is a viable method for amplifying vCJD PrP(Sc).
- Substrate choice, particularly PRNP codon 129 polymorphism, affects amplification efficiency.
- The developed method allows for the detection of sensitive PrP(Sc) forms, potentially aiding in early vCJD diagnosis.
