Rapid End-Point Quantitation of Prion Seeding Activity with Sensitivity Comparable to Bioassays

Abstract

A major problem for the effective diagnosis and management of prion diseases is the lack of rapid high-throughput assays to measure low levels of prions. Such measurements have typically required prolonged bioassays in animals. Highly sensitive, but generally non-quantitative, prion detection methods have been developed based on prions' ability to seed the conversion of normally soluble protease-sensitive forms of prion protein to protease-resistant and/or amyloid fibrillar forms. Here we describe an approach for estimating the relative amount of prions using a new prion seeding assay called real-time quaking induced conversion assay (RT-QuIC). The underlying reaction blends aspects of the previously described quaking-induced conversion (QuIC) and amyloid seeding assay (ASA) methods and involves prion-seeded conversion of the alpha helix-rich form of bacterially expressed recombinant PrPC to a beta sheet-rich amyloid fibrillar form. The RT-QuIC is as sensitive as the animal bioassay, but can be accomplished in 2 days or less. Analogous to end-point dilution animal bioassays, this approach involves testing of serial dilutions of samples and statistically estimating the seeding dose (SD) giving positive responses in 50% of replicate reactions (SD50). Brain tissue from 263K scrapie-affected hamsters gave SD50 values of 1011-1012/g, making the RT-QuIC similar in sensitivity to end-point dilution bioassays. Analysis of bioassay-positive nasal lavages from hamsters affected with transmissible mink encephalopathy gave SD50 values of 103.5–105.7/ml, showing that nasal cavities release substantial prion infectivity that can be rapidly detected. Cerebral spinal fluid from 263K scrapie-affected hamsters contained prion SD50 values of 102.0–102.9/ml. RT-QuIC assay also discriminated deer chronic wasting disease and sheep scrapie brain samples from normal control samples. In principle, end-point dilution quantitation can be applied to many types of prion and amyloid seeding assays. End point dilution RT-QuIC provides a sensitive, rapid, quantitative, and high throughput assay of prion seeding activity.

Cerebrospinal fluid real‐time quaking‐induced conversion is a robust and reliable test for sporadic creutzfeldt–jakob disease: An international study

“…Most laboratories used either a BMG LABTECH Optima or a BMG LABTECH Omega, whereas 1 laboratory used a Tecan Infinite F200PRO (Tecan Group, Männedorf, Switzerland). Nine laboratories used hamster full‐length (23–231) rPrP (supplied by Bristol Institute of Blood Sciences, Bristol, UK),8, 9 2 used human full‐length (23–231) rPrP (produced in‐house),7 and 1 used a hamster–sheep chimeric rPrP (hamster 14–126 residues followed by sheep residues 141‐234, produced according to previously reported conditions) 5…”

“…Shaking performed in a Thermomixer Comfort (Eppendorf, Hamburg, Germany) before being read in an Optima instrument.CSF = cerebrospinal fluid; Ham FL = hamster full‐length PrP (23–231)8, 9; Ham‐Sh chimeric = hamster residues 14–128 :sheep 141–234 5; Hum FL = human full‐length (23–231)—codon 129M6; PrP = prion protein; rfu = relative fluorescence units.…”

“…This technique uses recombinant PrP (rPrP) as a substrate, which is induced to aggregate by the addition of CSF containing PrP Sc . Thioflavin T (ThT) in the reaction binds to the aggregated PrP Sc , causing a change in the ThT emission spectrum, enabling the reaction to be monitored in real time 5, 6…”

Temporal Resolution of Misfolded Prion Protein Transport, Accumulation, Glial Activation, and Neuronal Death in the Retinas of Mice Inoculated with Scrapie

“…24,25 Briefly, rPrP composed of the Syrian hamster residues 90 to 231 in pET vector with N-terminal histidine sequence was transformed in to Escherichia coli Rosetta2(DE3) cells. The cultures were grown under continuous selection with kanamycin and chloramphenicol in the Lysogeny broth media.…”

“…The RT-QuIC assay was performed using previously published protocols from published reports 24,26,27 with slight modifications. The reaction mixtures consisted of final concentrations of 350 mmol/L NaCl, 0.1 mmol/L EDTA, 10 mmol/ L thioflavin T, 0.1 mg/mL rPrP, and 0.002% SDS in 1Â phosphate-buffered saline.…”