Prions are infectious agents involved in a number of degenerative brain diseases. These transmittable pathogens cause irregular folding of a brain protein in both animals and humans, which leads to brain damage and is fatal. Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE) and scrapie are a few examples of the neurodegenerative diseases caused by prions. In the past, lengthy bioassays were used to study prions in animal models over long periods of time, of between one and six months. As well as being time consuming, maintaining the infected animal for prolonged periods was a costly process.
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Prion Seeding Assay
A new prion seeding assay referred to as RealTime Quaking Induced Conversion Assay (RT-QuIC) has been developed by a team of scientists at Rocky Mountain Laboratories based in Hamilton, Montana. This assay provides end point quantitation for determining the levels of prions present in infected samples. Compared to prior techniques, this assay is faster and offers a higher throughput. It takes about 20 hours to complete the assay, which is as sensitive as using whole animal models and if not, more so.
Omega Series of Readers
The Omega series of plate readers designed by BMG LABTECH are capable of shaking and incubating microplates over extended time periods. Using a POLARstar Omega, the RT-QuIC samples were measured every 15 minutes for a period of 20 to 68 hours while being alternately shaken for a minute and rested for a minute. In order to regulate the periods of shaking and resting required for activating the assay, a software script was developed.
Figure 1. BMG LABTECH’s multidetection microplate reader FLUOstar Omega has shaking and incubation features that facilitate the RT-QuIC prion seeding assay. Image credits: BMG Labtech.
The FLUOstar Omega is a reliable, robust and effective microplate reader designed for prion seeding assays (Figure 1). The device offers excellent flexibility and performance for all life science and R&D applications. It saves significant amounts of time, allowing users to run their prion seeding assays in days rather than weeks. The FLUOstar Omega serves as an ideal platform for measuring prion seeding assays in a microplate format.
Rocky Mountain Laboratories has chosen the FLUOstar Omega as the reference microplate reader for developing the RT-QuIC assay, as well as for various peer-reviewed publications. In addition to its precision and robustness, the FLUOstar Omega is one of the only microplate readers designed to endure the long and high-speed shaking needed for prion seeding assays.
Principle of the Assay
The RT-QuIC assay integrates parts of both the original Quaking Induced Conversion (QuIC) assay and the amyloid seeding assay (ASA). It is used to estimate the relative quantity of prion seeding, measuring serial dilutions of samples and statistically estimating the seeding dose. To promote the misfolding of prion proteins, as observed in disease states, small quantities of infectious prions are introduced to normal prion proteins. The assay is quantitated by measuring serial dilutions of the samples and establishing the loss of seeding activity (SD50), which is the end point dilution.
Materials and Methods
For the experiment, the following materials were used:
- Nalgene Nunc plate sealer film
- Nalgene Nunc black with clear flat bottom 96 well plate
- FLUOstar Omega or POLARstar, BMG LABTECH, Ortenberg, Germany
Experimental Framework
For the entire 20 to 68 hour period, the Omega was set to incubate at a temperature of 42°C. A script was then generated to utilize two test run protocols: one that was set to shake the plate for one minute and rest for another minute and one that was set to obtain a fluorescence endpoint measurement via the bottom optic every 15 minutes. Thioflavin T (ThT), a fluorescent dye, was employed as a prion seeding marker. When this dye is added to recombinant prion proteins, it becomes integrated during polymerization, which leads to an increase in fluorescence over time. The instrument settings are described below:
- Emission filter: 480nm
- Excitation filter: 450nm
- Fluorescence endpoint- Bottom reading
- Gain: 2000
- Flashes: 20
- Shaking: 700RPM Double Orbital
Analysis
A Spearman-Kärber analysis, analagous to a bioassay’s lethal dose of 50% (LD50), was used to estimate a dilution or seeding dose at which half of the wells became ThT positive for each gram of tissue (SD50/g). For Spearman-Kärber analysis, a dilution series was chosen where at least one dilution gave 0% ThT positivity and one gave 100% ThT positivity.
Results and Discussion
The RT-QuIC assays were measured for 20 to 68 hours using a software script. The resulting data was collected as an average of relative fluorescence units across eight replicate wells. In the following figures, only data for every 45 minutes is shown.
Figure 2. RT-QuIC sensitivity: analysis of dilutions of a scrapie hamster brain homogenate stock 263K. The 50% seeding dose (SD50) is defined as the amount giving sufficiently enhanced ThT fluorescence in half of the replicate wells. In this case, the approximate SD50 was achieved with a 2 µl aliquot (the seed volume) of a 10E-9 dilution of the scrapie BH stock (dark blue line). Thus the Spearman-Kärber estimate for the SD50/2µL is 10E9.1 and for the SD50/g is 10E12. The log of this SD50 (~11) corresponded well with the log of the LD50 (~10) found in the bioassay. This figure was adapted from reference 2. Image credits: BMG Labtech.
The FLUOstar and the POLARstar Omega microplate readers are capable of shaking in a unique double orbital pattern, as well as orbital and linear patterns. For the incubation, a temperature of 42°C was maintained throughout the entire measurement time. Owing to the high quality design and manufacture of the Omega devices, researchers are able to perform continuous measurements for days at a time, without a temperature variations or a break in shaking. It is possible to obtain data without any interruption and output it to BMG LABTECH’s data analysis software (MARS) and Excel. Figures 2 and 3 demonstrate the RT-QuIC assay as it relates to hamster scrapie brain homogenates (BHs). Figure 2 used hamster BHs harvested 80 to 85 days following inoculation (DPI), while Figure 3 used hamster BHs harvested 10 days following inoculation.
The SD50/g of tissue for the 85 DPI samples (10E12) was found to be higher than for the 10 DPI (10E8.2), as predicted, due to the time for onset being longer. The RT-QuIC results were well correlated with the bioassay results. This confirms that the RT-QuIC assay may be regarded as a faster and higher throughput substitute for whole animal bioassays. The data also demonstrates that this assay can be used to examine animals as early as 10 days following inoculation.
Figure 3. RT-QuIC end-point dilution analysis of three 263K-inoculated preclinical 10 days post injection hamster BHs. In this case, the approximate SD50 was achieved with a 2 µl aliquot (the seed volume) of a 10E−5 dilution of the scrapie 10dpi 263K BH stock (green line). This gave an SD50/2µL of 10E5.5 and an SD50/g of 10E8.2. This figure was adapted from reference 2. Image credits: BMG Labtech.
Conclusion
The RT-QuIC assay and a microplate reader can be used to measure prion seeding at a faster speed and higher throughput. A few spongiform encephalopathies that have been shown to work using RT-QuIC include CJD, BSE, sheep and hamster scrapie and deer chronic wasting disease. The RT-QuIC assay is not only faster, but also more sensitive when compared to bioassays used in the past. BMG LABTECH’s Omega series of plate readers are robust and functional enough to endure the many days of shaking at high speeds needed for a prion seeding assay.
References
- https://www.cdc.gov/
- Wilham, J. M., et al. (2010) Rapid End-Point Quantitation of Prion Seeding Activity with Sensitivity Comparable to Bioassays in PLoS Pathog 6 (12): e1001217. doi:10.1371/journal.ppat.1001217
- Atarashi R, et al. 2008. Simplified ultrasensitive prion detection by recombinant PrP conversion with shaking. Nat Methods 5: 211–212.
- Colby DW, et al. 2007. Prion detection by an amyloid seeding assay. Proc Natl Acad Sci U S A 104 : 20914–20919.
- Atarashi, R. et al. 2011. Real-time Quaking Induced Conversion. Prion 5 (3). PMC3226039.
About BMG Labtech
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