Under certain conditions the normal prion protein in the presence of the aggregated conformer undergoes conversion and produces enough aggregated prion protein to be readily detectable. One such assay relies on the recombinant prion protein expressed in bacteria as a substrate for conversion. This method is called the quaking induced conversion assay (QuIC). Using thioflavin T – which florescences on binding aggregated protein – the conversion of the recombinant can be followed in real-time (RT-QuIC).RT-QuIC using NIBSC control and sporadic CJD brain homogenates
We have generated several recombinant prion proteins – Syrian hamster and hamster/sheep chimera – that amplify PrPSc derived from both sporadic and variant CJD tissue. For the hamster/sheep chimera the sensitivity is greater with sporadic CJD-derived PrPSc seed than with vCJD-derived material. Optimising the RT-QuIC assay for the amplification of vCJD is our current focus.
Disease diagnosis is becoming increasingly complex. There is significant focus on early detection of disease – before clinical onset – using biomarkers which show changes in levels or location of normal cellular proteins/nucleic acid.
Biomarkers are currently used to detect cancers and complex neurological disorders. 14-3-3 gamma is a diagnostic marker for sporadic CJD and is one of the World Health Organisation’s (WHO) criteria for diagnosis.
Recombinant 14-3-3 gamma is used to diagnose sporadic CJD and other neurological conditions. Diagnosing sporadic CJD can only be confirmed by biopsy, although indicators of disease are known – such as the presence of 14-3-3 gamma in cerebrospinal fluid (CSF) samples. A positive test for 14-3-3, in combination with other neurological signs typical of disease, is considered indicative of sporadic CJD. We analyse CSF samples for the presence of 14-3-3 by western blot analysis. The human 14-3-3 gamma is overexpressed in E.coli and purified to apparent homogeneity. This recombinant is used as a standard to estimate the concentration of 14-3-3 in patient samples.