Superparamagnetic Nanoparticle Capture of Prions for Amplification

Abstract

Prion diseases are associated with the presence of PrPSc , a disease-associated misfolded conformer of the prion protein. We report that superparamagnetic nanoparticles bind PrP Sc molecules efficiently and specifically, permitting magnetic separation of prions from a sample mixture. Captured PrP Sc molecules retain the activity to seed protein misfolding cyclic amplification (PMCA) reactions, enabling the rapid concentration of dilute prions to improve detection. Furthermore, superparamagnetic nanoparticles clear contaminated solutions of PrP Sc . Our findings suggest that coupling magnetic nanoparticle capture with PMCA could accelerate and improve prion detection. Magnetic nanoparticles may also be useful for developing a nontoxic prion decontamination method for biologically derived products.Bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, and other prion diseases are caused by an infectious agent that contains PrP Sc , a misfolded conformer of the normal cellular prion protein (PrP C ) (24). Low-abundance sources of prions, such as blood, may still transmit disease (17,23). Prion diseases currently have no therapy, nor can prions be specifically removed from contaminated material.Inoculation bioassay serves as the gold standard for specific detection of prion infectivity. For sensitive detection, protein misfolding cyclic amplification (PMCA) has emerged as a rapid alternative to bioassay. PMCA exploits prion multiplication mechanisms to amplify PrP Sc in vitro using PrP C substrate (1, 5). Analogous to amplification of DNA sequence template by PCR, PrP Sc template seeds the conversion of PrP C substrate in PMCA, resulting in propagation and amplification of the PrP Sc conformation. Each serial PMCA round increases the detection sensitivity exponentially but requires ϳ72 h (7). The application of PMCA is also limited by prion propagation inhibitors present in blood and other biological solutions (6). An effective method to concentrate prions would improve subsequent PMCA sensitivity and utility.Nanotechnology presents many opportunities for fine control of molecular events. Certain iron oxide crystals less than ϳ25 nm in diameter exhibit superparamagnetism, with a net magnetization only occurring in the presence of an external magnetic field (15). MagnaBind and Dynal superparamagnetic beads contain many iron oxide crystals, dispersed such that no permanent magnetic order can form. This enables the whole particles to be superparamagnetic, allowing them to be rapidly attracted to a magnet and to lose magnetic interactions upon removal of the magnet (28). In molecular biology, superparamagnetic beads are often conjugated to specifically bind a target molecule.Using superparamagnetic nanoparticles, we have identified a novel binding interaction with PrPSc . Magnetic capture of PrP Sc may be applied to prion detection and prion decontamination. MATERIALS AND METHODSPreparation of scrapie-infected and uninfected brain homogenate. CD-1 mouse (prion strains RML, Me7, and 301C) and Syrian hamster (prion ...

Enhanced prion detection in biological samples by magnetic particle extraction and real-time quaking-induced conversion

“…The propensity for prions to bind to metal surfaces has been well known dating to the iatrogenic transmission of CJD through contaminated stainless steel surgical instruments, even after sterilization (Gibbs et al, 1994;Bernoulli et al, 1977). The present work builds on that of other prion researchers who have employed antibody-conjugated and untreated steel or iron beads for prion activity amplification (Edgeworth et al, 2011;Orrú et al, 2011;Miller & Supattapone, 2011). It should also be noted that other investigators have observed that the use of protein coating and PrP-specific antibodies on beads may inhibit binding and prion amplification (Müller-Schiffmann et al, 2009).…”

“…Metal-binding properties have been exploited to detect prions in brain using PMCA and in blood using enzyme-linked immunosorbent assay (ELISA) with or without cell-culture amplification (Miller & Supattapone, 2011;Lim et al, 2015;Edgeworth et al, 2011). In addition, Orrú et al (2011) demonstrated that antibody-conjugated metal beads incubated with prion-positive brain and blood could bind prion-seeding activity detectable by RT-QuIC.…”

Advances in the Treatment of Neurodegenerative Disorders Employing Nanoparticles

“…Other methods, such as sodium hydroxide, sodium hypochlorite, and phosphotungstic acid treatments, destroy or remove prions but also damage the material of interest. In contrast, magnetic nanoparticles capture PrPSc with specificity [149]. Obviously, the developing of this technique can be applied also to prion detection in biological samples.…”

“…In this contribution, there were published different strategies using different aspects of the nanotechnology with quantum dots [147,148] and suparamagnetic nanoparticles [149].…”

The Tyr-Tyr-Arg Prion-Specific Epitope: Update and Context

“…The speci fi c immunoconcentration of PrP C and PrP Sc can be done by using Ab bound to paramagnetic beads or columns. PrP C and PrP Sc can be brought down together using an anti-PrP Ab, or PrP Sc can be speci fi cally concentrated from dilute biological fl uids using PrP Sc -speci fi c Abs (Paramithiotis et al 2003 ;Miller and Supattapone 2011 ;Orru et al 2011 ) .…”