Amplification of disease associated amyloid protein
Amyloid amplification assays were first developed for the diagnosis of TSEs. These assays increase the levels of the disease associated misfolded protein to levels detectable by conventional methods.
The protein-misfolded cyclic amplification assay (PMCA) utilises cycles of sonication and rest in order to convert normally folded prion protein substrate in brain homogenate to its disease associated conformation. The presence of disease associated prion protein is confirmed by western blot analysis following treatment with proteinase K. This method allows for the detection of variant CJD-specific material and is sufficiently sensitive to detect disease marker in blood and urine.
A modification of this assay, real-time quaking induced conversion assay (RT-QuIC), utilises bacterially expressed recombinant protein as a substrate, with the increase in disease associated material being monitored in real time using a fluorescent marker, Thioflavin T. This method improved detection for sporadic CJD, however it was unable to detect variant CJD associated material at similar efficiencies.
Improving RT-QuIC for the detection of sporadic CJD
The Centre has generated numerous recombinant prion proteins from several species (including hamster and bank vole) in full length (amino acids 20-231) and truncated forms (90-231). We continue to assess these materials against both variant and sporadic CJD reference reagents. We find that these recombinant materials give strong responses for amplification of sporadic CJD, however they amplify poorly for variant CJD.
RT-QuIC applied to diverse amyloid disorders
RT-QuIC is under development for the amplification and detection of disease associated material in more common amyloid diseases by a number of laboratories internationally. The Centre is working towards establishing the utility of the RT-QuIC method for the detection of amyloid material associated with common neurodegenerative disorders, with specific interest in Parkinson’s and Alzheimer’s disease.
The Centre has successfully generated a library of recombinant material, including alpha synuclein protein carrying common human genetic polymorphisms associated with Parkinson’s disease. We have used these proteins to improve the performance of the RT-QuIC, improving sensitivity and the capacity to differentiate samples from commonly misdiagnosed neurodegenerative conditions.
Recombinant proteins as controls for disease biomarker analysis
With the growing complexity of disease diagnosis, there is an increasing focus on early detection of disease, ideally before the onset of clinical symptoms. Biomarkers which reveal a change in the level or localisation of normal biological components have formed a cornerstone of this new endeavor.
Diagnosis of sporadic CJD can presently only be confirmed by biopsy. There are known indicators of disease however, and a test positive for the presence of one such indicator, 14-3-3 gamma protein in cerebrospinal fluid, is one of the WHO criteria for diagnosis. A positive test for 14-3-3 gamma is considered indicative of sporadic CJD when presented in conjunction with other neurological symptoms typical of the disease.
The Centre works to analyse cerebrospinal fluid samples using recombinant human 14-3-3 gamma protein overexpressed in E. coli and purified using HPLC methods. Cerebrospinal fluid is screened for 14-3-3 gamma content using western blot analysis, and our 14-3-3 gamma standard is used to assess the concentration of the marker in patient samples.