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 PrP^Sc 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 PrP^Sc in peripheral tissues and brain of vCJD patients, based on the differential extraction of PrP^Sc 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 PrP^Sc infected and non-infected tissues. The assay has a detection limit of 10 pg PrP, is robust and could be automated.