Prion Disease Blood Test Using Immunoprecipitation and Improved Quaking-Induced Conversion

Abstract

A key challenge in managing transmissible spongiform encephalopathies (TSEs) or prion diseases in medicine, agriculture, and wildlife biology is the development of practical tests for prions that are at or below infectious levels. Of particular interest are tests capable of detecting prions in blood components such as plasma, but blood typically has extremely low prion concentrations and contains inhibitors of the most sensitive prion tests. One of the latter tests is quaking-induced conversion (QuIC), which can be as sensitive as in vivo bioassays, but much more rapid, higher throughput, and less expensive. Now we have integrated antibody 15B3-based immunoprecipitation with QuIC reactions to increase sensitivity and isolate prions from inhibitors such as those in plasma samples. Coupling of immunoprecipitation and an improved real-time QuIC reaction dramatically enhanced detection of variant Creutzfeldt-Jakob disease (vCJD) brain tissue diluted into human plasma. Dilutions of 1014-fold, containing ~2 attogram (ag) per ml of proteinase K-resistant prion protein, were readily detected, indicating ~10,000-fold greater sensitivity for vCJD brain than has previously been reported. We also discriminated between plasma and serum samples from scrapie-infected and uninfected hamsters, even in early preclinical stages. This combined assay, which we call “enhanced QuIC” (eQuIC), markedly improves prospects for routine detection of low levels of prions in tissues, fluids, or environmental samples.

First Demonstration of Transmissible Spongiform Encephalopathy-associated Prion Protein (PrPTSE) in Extracellular Vesicles from Plasma of Mice Infected with Mouse-adapted Variant Creutzfeldt-Jakob Disease by in Vitro Amplification

“…can reliably identify preclinical cases to minimize the risk of iatrogenic disease transmission via blood-derived products. Experimental detection of PrP TSE in animal cerebrospinal fluid (CSF) (29), whole blood (21,22), plasma (27)(28)(29)(30)71), buffy coat (24 -26), and urine (74) and in human CSF (12,75), whole blood (6 -8), and urine (76,77) has been achieved by different methods that rely on the concentration or amplification techniques to bring PrP TSE levels to the detection threshold of biochemical assays. Although the presence of PrP TSE in blood exosomes has been suggested previously (48), biochemical detection has been complicated by the low levels of PrP TSE in blood and the concomitantly large volumes required for exosome isolation by standard methods.…”

“…PrP TSE has been successfully detected in the following: in whole blood of experimentally infected mice and hamsters, in sheep with natural scrapie and experimental BSE, and in white-tailed deer afflicted with CWD (21)(22)(23); in buffy coats of hamsters and sheep experimentally infected with scrapie (24 -26), as well as in plasma of mice and hamsters with scrapie; and in sheep and white-tailed deer naturally and experimentally infected with scrapie and CWD, respectively (27)(28)(29)(30). However, it is still not fully understood in what blood component TSE infectivity and/or PrP TSE reside and whether and how blood contributes to the spread of the disease from the periphery to the brain.…”

Factors intrinsic and extrinsic to blood hamper the development of a routine blood test for human prion diseases

“…It is thought that this may be due to condition-dependent factors, such as differences in the propensity to form an amyloidogenic precursor, or an intermediate that may be totally different from the native rPrP C that interacts more favourably and efficiently with prion seeds. Alternatively, it may be due to the occurrence of off-pathway changes that may promote spontaneous, prion-independent fibril formation (false positives) or remove rPrP C from the assembly component pool (Orrú et al, 2011).…”

“…Other researchers have attempted a modified PMCA approach, known as enhanced quaking-induced conversion (e-QuIC) (Orrú et al, 2011). This approach combines QuIC, which can be as sensitive as an in vivo bioassay and in which prions induce the polymerization of recombinant PrP C (rPrP C ) into amyloid fibrils by applying cycles of shaking and rest (Atarashi et al, 2008;Orrú et al, 2009), with specific IP of PrP Sc using the 15B3 antibody (Korth et al, 1997;Biasini et al, 2008Biasini et al, , 2009.…”

A new diagnostic test for Creutzfeldt-Jakob disease: Real-time quaking-induced conversion (RT-QuIC)

“…However, ongoing validation studies have demonstrated that the outcomes of RT-QuIC reactions depend on the exact conditions employed (biochemical, chemical and physical), as well as the nature of the sample and recombinant substrate. The protocol under evaluation by the National Microbiology Laboratory has been tested extensively for the detection of sporadic CJD (10,11,15,16 ). Using this protocol the Prion Laboratory Section achieved 100% sensitivity and 100% specificity in the evaluation of an external sporadic CJD infected CSF proficiency panel.…”