Real-time quaking-induced conversion (RT-QuIC) allows the amplification of miniscule amounts of scrapie prion protein (PrPSc). Recent studies applied the RT-QuIC methodology to cerebrospinal fluid (CSF) for diagnosing human prion diseases. However, to date, there has not been a formal multi-centre assessment of the reproducibility, validity and stability of RT-QuIC in this context, an indispensable step for establishment as a diagnostic test in clinical practice. In the present study, we analysed CSF from 110 prion disease patients and 400 control patients using the RT-QuIC method under various conditions. In addition, “blinded” ring trials between different participating sites were performed to estimate reproducibility. Using the previously established cut-off of 10,000 relative fluorescence units (rfu), we obtained a sensitivity of 85 % and a specificity of 99 %. The multi-centre inter-laboratory reproducibility of RT-QuIC revealed a Fleiss’ kappa value of 0.83 (95 % CI: 0.40–1.00) indicating an almost perfect agreement. Moreover, we investigated the impact of short-term CSF storage at different temperatures, long-term storage, repeated freezing and thawing cycles and the contamination of CSF with blood on the RT-QuIC seeding response. Our data indicated that the PrPSc seed in CSF is stable to any type of storage condition but sensitive to contaminations with blood (>1250 erythrocytes/μL), which results in a false negative RT-QuIC response. Fresh blood-contaminated samples (3 days) can be rescued by removal of erythrocytes. The present study underlines the reproducibility and high stability of RT-QuIC across various CSF storage conditions with a remarkable sensitivity and specificity, suggesting RT-QuIC as an innovative and robust diagnostic method.Electronic supplementary materialThe online version of this article (doi:10.1007/s12035-015-9133-2) contains supplementary material, which is available to authorized users.

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.

Evidence is accumulating that intracellular animal lectins play important roles in quality control and glycoprotein sorting along the secretory pathway. Calnexin and calreticulin in conjunction with associated chaperones promote correct folding and oligomerization of many glycoproteins in the endoplasmic reticulum (ER). The mannose lectin ERGIC-53 operates as a cargo receptor in transport of glycoproteins from ER to Golgi and the homologous lectin VIP36 may operate in quality control of glycosylation in the Golgi. Exit from the Golgi of lysosomal hydrolases to endosomes requires mannose 6-phosphate receptors and exit to the apical plasma membrane may also involve traffic lectins. Here we discuss the features of these lectins and their role in glycoprotein traffic in the secretory pathway. ß

The absence of infectivity‐associated, protease‐resistant prion protein (PrPSc) in the brains of spontaneously sick transgenic (Tg) mice overexpressing PrP linked to Gerstmann–Sträussler Scheinker syndrome, and the failure of gene‐targeted mice expressing such PrP to develop disease spontaneously, challenged the concept that mutant PrP expression led to spontaneous prion production. Here, we demonstrate that disease in overexpressor Tg mice is associated with accumulation of protease‐sensitive aggregates of mutant PrP that can be immunoprecipitated by the PrPSc‐specific monoclonal antibody designated 15B3. Whereas Tg mice expressing multiple transgenes exhibited accelerated disease when inoculated with disease‐associated mutant PrP, Tg mice expressing mutant PrP at low levels failed to develop disease either spontaneously or following inoculation. These studies indicate that inoculated mutant PrP from diseased mice promotes the aggregation and accumulation of pre‐existing pathological forms of mutant PrP produced as a result of transgene overexpression. Thus, while pathological mutant PrP possesses a subset of PrPSc characteristics, we now show that the attribute of prion transmission suggested by previous studies is more accurately characterized as disease acceleration.

The development of in vitro amplification systems allows detecting femtomolar amounts of prion protein scrapie (PrPSc) in human cerebrospinal fluid (CSF). We performed a CSF study to determine the effects of prion disease type, codon 129 genotype, PrPSc type, and other disease-related factors on the real-time quaking-induced conversion (RT-QuIC) response. We analyzed times to 10,000 relative fluorescence units, areas under the curve and the signal maximum of RT-QuIC response as seeding parameters of interest. Interestingly, type of prion disease (sporadic vs. genetic) and the PRNP mutation (E200K vs. V210I and FFI), codon 129 genotype, and PrPSc type affected RT-QuIC response. In genetic forms, type of mutation showed the strongest effect on the observed outcome variables. In sporadic CJD, MM1 patients displayed a higher RT-QuIC signal maximum compared to MV1 and VV1. Age and gender were not associated with RT-QuIC signal, but patients with a short disease course showed a higher seeding efficiency of the RT-QuIC response. This study demonstrated that PrPSc characteristics in the CSF of human prion disease patients are associated with disease subtypes and rate of decline as defined by disease duration.Electronic supplementary materialThe online version of this article (doi:10.1007/s12035-014-8709-6) contains supplementary material, which is available to authorized users.