Recombinant prion protein for high-throughput prion diagnosis
Bayerische Patentallianz GmbH
Transmissible spongiform encephalopathies (TSEs) like Creutzfeldt-Jakob disease (CJD) and Bovine spongiform encephalopathy (BSE), also known as „prion diseases“, are a group of transmissible neurodegenerative diseases
affecting primarily the central nervous system (CNS) of many animals, including humans. Transmission occurs by blood transfusion, transplantation and the food chain. Accordingly there is a strong need for diagnostic methods to
detect the infectious agent, an abnormal, misfolded form of the prion protein (PrPSc), especially in order to discriminate the prion cases from other neurodegenerative diseases.
Problems in prion diagnosis are due to the low concentration of PrPSc in easily accessible specimens such as cerebrospinal fluid (CSF) and blood. Therefore, a novel amplification system was developed, termed quaking-induced conversion
(QuIC), based on the knowledge that PrPSc can propagate autocatalytically in vitro by converting cellular prion protein (PrPC) and recombinant prion protein (rPrP) to pathogenic forms. A novel real-time QuIC (RT-QuIC) uses an
auto-read set-up to monitor the prion propagating procedure with fluorescence so that testing of multiple samples on one 96-well plate is now possible. However, it was found that this method leads to false-negative and falsepositive results and appears to be quite inefficient. Therefore, a need remains for a rapid and reliable diagnostic method for prion proteins.
It could be shown that the problems concerning specificity and efficiency of RT-QuIC result from insufficiently purified rPrP, which means that the key issue of the meanwhile widely accepted RT-QuIC-technique is to choose a correct
way to purify reliable rPrP, with lower contamination and higher activity for the RT-QuIC system.
The present invention uses immobilized metal ion affinity chromatography (IMAC) to purifiy human rPrP and a novel elution method based on imidazole for harvesting the protein binding to the Ni2+ column. One of the elution peaks
was selected as substrate for RT-QuIC due to its high activity and stability, which opened up impressive new opportunities in the field of prion disease diagnosis.
The present invention shows several advantages concerning the diagnosis of prion proteins
- “ready-to-use protein”
- suitable for prion diagnosis in brain, CSF and blood
- increase of RT-QuIC efficiency
- decrease of test time
- 100% specificity
- production of 200 mg rPrP per month by one operator possible a 20.000 diagnostic tests
- fast and uncomplicated tests for suspected prion disease or common screenings in human medicine, agriculture, food safety import and export etc.
- „ready-to use protein“
- Proven effect