PAD-Beads enrichment enhances detection of PrPSc using real-time quaking-induced conversion

Soyoun Hwang,Eric M Nicholson,Rohana P Dassanayake

doi:10.1186/s13104-019-4842-7

Abstract

ObjectiveScrapie is a transmissible spongiform encephalopathy (TSE) that naturally occurs in sheep and goats. This fatal neurodegenerative disease results from misfolding of the normal cellular prion protein (PrPC) to a pathogenic prion protein form (PrPSc). This pathogenic form, PrPSc, accumulates in the brain and lymphoid tissues. The presence of PrPSc can be detected by an in vitro conversion assay known as real-time quaking induced conversion (RT-QuIC). RT-QuIC has been used to detect PrPSc in a variety of biological tissues from brains to fluids. While this technique is both rapid and sensitive, enhancing the detection of prions would be valuable in the diagnostic laboratories.ResultsIn this study, we assessed whether PrPSc detection sensitivity of RT-QuIC can be increased by enriching PrPSc in scrapie tissue homogenates using commercially available aggregated protein binding ligands coated magnetic beads (PAD-Beads). Coupling of RT-QuIC to PAD-Beads based cleanup allowed detection of PrPSc rapidly and without dilution of scrapie sheep brain homogenates prior to RT-QuIC. The PAD-Beads sample pretreatment step prior to RT-QuIC is a useful enhancement in the diagnosis of TSEs.

Highlights

Mammalian prion diseases include human Creutzfeldt– Jakob disease (CJD), bovine spongiform encephalopathy (BSE), sheep scrapie, and cervid chronic wasting disease (CWD). These diseases are fatal neurologic diseases known as transmissible spongiform encephalopathies (TSEs), and they result from the misfolding of the normal cellular prion protein (PrPC) into a pathogenic form (pathogenic prion protein (PrPSc)) that accumulates primarily in the central nervous system [1–4]
RT‐QuIC detection of PAD‐Beads captured scrapie prions To evaluate the efficacy of PAD-Beads based enrichment for the purposes of real-time quaking induced conversion (RT-QuIC), reactions containing recombinant bank vole prion protein (BV recombinant prion protein (rPrPC)) were seeded with different dilutions of PAD-Beads eluate for comparison with that of the directly diluted sheep brain homogenate and monitored for increased thioflavin T (ThT) fluorescence
With the amplification of sensitivity afforded by the RT-QuIC technique, early diagnosis is becoming an increasingly feasible proposition since the technique amplifies the pathogenic form of prion protein in biological tissue or fluid samples

Summary

Results

RT‐QuIC detection of PAD‐Beads captured scrapie prions To evaluate the efficacy of PAD-Beads based enrichment for the purposes of RT-QuIC, reactions containing recombinant bank vole prion protein (BV rPrPC) were seeded with different dilutions of PAD-Beads eluate for comparison with that of the directly diluted sheep brain homogenate and monitored for increased ThT fluorescence. RT-QuIC reactions containing bank vole substrate and 300 mM NaCl were seeded with brain stock solution (Fig. 1a, c) or the PAD-Beads eluate (Fig. 1b,d) and dilutions from 10−1 to 10−7 of brain homogenate from two sheep (#1 and #2) positive for scrapie Both reactions seeded with brain stock and PAD-Beads eluted brain showed fibril formations as monitored by ThT fluorescence. RT-QuIC reactions seeded with PAD-Beads eluted in a 100 μl volume but bound to twice the volume of PAD-Beads used in the standard protocol provided a similar reduction in lag time to the onset of ThT fluorescence to that observed for the standard elution volume In this case, all brain dilutions up to 10−7 have higher seeding activity showing lag time shorter than 40 h compared to the reactions seeded with eluent from the lower amount of beads. Increased bead volume resulted in higher sensitivity indicating that for these samples and the lower amount of beads used the binding capacity of the beads was exceeded

Introduction

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Discussion and conclusions

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