An immunoassay for the pathological form of the prion protein based on denaturation and time resolved fluorometry

Concern about the possible secondary spread of variant Creutzfeldt–Jakob disease (vCJD) through blood transfusion and blood products has increased the need for a sensitive and rapid test for the identification of PrPSc in specimens collected non-invasively from living persons. Furthermore, an accurate estimate of the prevalence of pre-clinical vCJD in the British population would be possible if there were such a test that could be applied to specimens available readily (e.g. blood and urine). As a first step towards that goal, we have developed a simple and sensitive test for the detection of PrPSc in peripheral tissues and brain of vCJD patients, based on the differential extraction of PrPSc with guanidine hydrochloride. The prion protein (PrP) isoforms are extracted sequentially from homogenized tissue by applying two different concentrations of this chaotropic agent. Each extraction yields a fraction of the PrP isoforms with different solubilities in guanidine hydrochloride. Quantitation of the two fractions (relatively insoluble or relatively soluble) using time resolved fluorescence (DELFIA) as a reporter system allows differentiation between PrPSc infected and non-infected tissues. The assay has a detection limit of 10 pg PrP, is robust and could be automated.