Prion Research: Recent Developments
As you might be aware, BMG LABTECH microplate readers have played a central role in prion research. Specifically, the real-time quaking induced conversion assay (RT-QuIC) was developed using BMG LABTECH’s Omega. This has become a standard assay for detection of PrPSc from a variety of species, including humans, from various tissues and excretions. This assay takes advantage of the solid construction of BMG microplate readers to perform cycling of shaking on and off for an extended time while periodically reading the plate to monitor changes in fluorescent signal as Thifolavin T is incorporated into PrPSc aggregates. The CLARIOstar®, in addition to the, Omega has been successfully used for this application and BMG LABTECH continues to respond to suggestions from the research community to find ways to make performance of the RT-QuIC assay easier to perform with our readers.
Of course, most our readers are not one-trick ponies and can perform multiple different types of detection. This is highlighted in some of the recent reports within the field of prion research.
The first paper I would like to discuss is entitled: ‘Structural attributes of mammalian prion infectivity: Insights from studies with synthetic prions’ which was written by researchers from Case Western in Cleveland, USA. This paper employs a technique called Conformational Dependent Immunoassay or CDI. This assay takes advantage of the epitope specificity that is possible for monoclonal antibodies and the fact that different forms of PrP contain distinct epitopes that can be recognized by these antibodies. Using two antibodies designed to specifically recognize two different epitopes of PrP, the CDI becomes a fairly straightforward sandwich immunoassay in which samples are bound to plates using the capture antibody and subsequently incubated with detection antibody. Detection, in this case, is achieved through the antibody being labeled with a lanthanide to enable TRF detection. Li, et. al used BMG Labtech's PHERAstar® for detection of their CDI.
Li et al. used CDI as part of their investigation into what structural features in misfolded proteins are important for infectivity. They compared the susceptibility of infective (rPrP-resRNA) and a non-infective (rPrP-resRNA-low) versions to denaturation by guanidinium hydrochloride (GdnHCl). In this CDI the only time both antibodies will bind is when both epitopes are exposed by unfolding. These tests showed a large difference between the 2 forms with regard to stability in the presence of GdnHCl. This difference correlates with the other structural differences observed between these two forms of differing infectivity. The authors expect that their results will assist in construction of high-resolution models for infectious prions.
The second paper: ‘A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization’ looked at the progressive binding of PrP, focusing on the initial dimerization event. To achieve their goal Wüsten et al. used an assay based on Gaussia luciferase (Glu) complementation which they call: bioluminescent prion assay (BPA). In BPA, 2 forms of PrP were expressed in RK13 cells. One form was tagged with the N-terminal portion GLuc the other with the C-terminal portion. When the 2 forms of PrP are brought together by the dimerization event the 2 portions of the GLuc are also brought together. The result is a complete GLuc that can now function to produce light in the presence of the appropriate substrate coelenterazine.
Wüsten et al. used this assay to develop a compound screen to identify small molecules that inhibit PrP dimerization. From the screen 13 candidates were selected that reduce dimerization dependent luciferase signal by 60-80%.
Additional characterization of JTC-801 showed that is able to disrupt dimerization at concentrations that did not affect cell survival. Furthermore, it decreased PrPSc in 2 other cell lines based on a separate biochemical assay. The authors conclude that the future from applying BPA to discover species-specific inhibitors of PrP dimerization is bright (pun definitely intended).
BMG LABTECH is always proud to be associated with outstanding research such as prion research! If you would like to know more visit us at www.bmglabtech.com. Or if you are in San Diego for SfN please come see us!