In vitro detection of prions

A method for the amplification of prion disease-associated isoform of prion protein (PrPD) in a sample comprising the steps of:

• providing a sample having PrPD or a sample to be screened for the presence of PrPD;
• performing lipase treatment of the sample;
• contacting the lipase-treated sample with magnetic iron oxide beads (IOBs), whereby the IOBs bind PrPD in the sample;
• recovering the IOBs from the lipase-treated sample;
• providing a reaction mixture comprising an excess of normal, non-pathogenic isoform of prion protein (PrPC);
• combining the reaction mix and the recovered IOBs;
• incubating the reaction mixture at about 55° C. or higher under conditions effective to cause misfolding or aggregation of the PrPC in the combined reaction mixture;
• disaggregating any aggregates of PrPD formed during the incubating step;
• and
• repeating the incubating and disaggregating steps one or more times to produce an amplified PrPD in the reaction mixture.

A method for the detection of a prion disease-associated isoform of prion protein (PrPD) in a blood sample comprising the steps of:

• collecting a whole blood sample to be processed for the screening of PrPD;
• performing lipase treatment of the sample;
• performing metal bead extraction on the lipase-treated sample using magnetic iron oxide beads (IOBs);
• recovering the bead fraction of the sample;
• amplifying the PrPD in the sample using a plurality of rounds of protein misfolding cyclic amplification (PMCA) reactions with substrate;
• combining the amplified sample with Thioflavin T;
• re-amplifying the sample using a real-time quaking-induced conversion assay (RT-QuIC) reaction at about 55° C. or higher;
• and
• detecting the presence of PrPD in the whole blood sample by measuring the resulting fluorescence in the sample.

A method for the detection of a prion disease-associated isoform of prion protein (PrPD) in a blood sample comprising the steps of:

• providing a buffy coat cell fraction of a blood sample;
• performing lipase treatment of the buffy coat cell fraction;
• contacting the lipase-treated buffy coat cell fraction with magnetic iron oxide beads (IOBs);
• recovering the IOB fraction of the lipase-treated sample;
• amplifying the PrPD in the sample using a plurality of rounds of protein misfolding cyclic amplification (PMCA) reactions;
• combining the amplified sample with Thioflavin T;
• re-amplifying the sample using a real-time quaking-induced conversion assay (RT-QuIC) reaction at about 55° C. or higher;
• and
• detecting the presence of PrPD in the sample by measuring the resulting fluorescence in the sample.