A novel real-time ultrasonic method for prion protein detection using plasminogen as a capture molecule

Carmen Negredo,Eoin Monks,Torres Sweeney

doi:10.1186/1472-6750-7-43

Abstract

BackgroundHigh resolution ultrasonography (HR-US) can monitor the molecular changes and biochemical interactions between proteins in real-time. The aim of this study was to use HR-US to characterize the real-time interactions between plasminogen coated beads and PrPSc and to determine if this approach could be applied to the identification of animals affected by prion diseases. Plasminogen, immobilized to beads, was used as a capturing tool for PrPSc in brain homogenates from scrapie affected sheep and the binding reaction was monitored in real-time in an ultrasonic cell.ResultsChanges in the ultrasonic parameters suggested that three processes occurred during the incubation: binding, protein-protein network formation and precipitation and that these processes occurred in a concentration dependent manner. Conversely, when homogenates from normal sheep were similarly examined, no evidence for the occurrence of these processes was found indicating the specificity of the interaction between the plasminogen coated beads and PrPSc.ConclusionThese results indicate firstly, that the plasminogen coated beads binded selectively to PrPSc and secondly, that a HR-US system can discriminate between scrapie affected and non-affected samples and thus has potential as a tool for the rapid diagnosis for prion diseases. This approach has the significant advantage of not requiring a proteinase K pre-digestion step, which is routinely used in current PrPSc detection assays.

Highlights

High resolution ultrasonography (HR-US) can monitor the molecular changes and biochemical interactions between proteins in real-time
Prion diseases such as CJD in humans, BSE in cattle and scrapie in sheep are a group of neurodegenerative disorders, which are characterised by the accumulation in the central nervous system of the protease resistant form (PrPSc) of a host-coded membrane glycoprotein (PrPc) [1]
While PrPc exist as a monomer and it is rapidly degraded by proteinase K (PK), the infectious isoform PrPSc, forms detergent-insoluble aggregates and displays a higher resistance to degradation with PK [4,5]

Summary

Introduction

High resolution ultrasonography (HR-US) can monitor the molecular changes and biochemical interactions between proteins in real-time. Plasminogen, immobilized to beads, was used as a capturing tool for PrPSc in brain homogenates from scrapie affected sheep and the binding reaction was monitored in real-time in an ultrasonic cell. Prion diseases such as CJD in humans, BSE in cattle and scrapie in sheep are a group of neurodegenerative disorders, which are characterised by the accumulation in the central nervous system of the protease resistant form (PrPSc) of a host-coded membrane glycoprotein (PrPc) [1]. The transformation of PrPc into PrPSc implies a conformational change from a mainly alpha helical form into a beta sheet rich structure [2,3] This conformational difference is responsible for the distinct physicochemical properties of both isoforms. As antibodies used on current validated assays are not able to differentiate between PrPSc and PrPc, these diagnostic procedures rely on the (page number not for citation purposes)

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