Thursday, February 21, 2013

Applications Thursday - Faster, More Sensitive Prion Seeding Assay

A newly developed prion seeding assay called Real-Time Quaking Induced Conversion assay (RT-QuIC) now permits researchers to measure prion seeding at lower levels and in less than a few days in microplates. This is in contrast to standard animal bioassays that are sensitive enough, but are more expensive and much slower (they can years versus only days). RT-QuIC is an improvement on the previously employed methods of quaking-induced conversion (QuIC) (Atarashi 2008) and amyloid seeding assay (ASA) (Colby 2007).

In order to better understand the spread of various infectious prion diseases (Transmissible Spongiform Encephalopathies ), including those that affect humans (Creutzfeldt-Jakob disease (CJD)) or animals (i.e. Scrapie in sheep or Chronic Wasting Disease in deer), researchers need a better and faster way to quantitate the infectivity or number of prions in the infected host. RT-QuIC, when used on BMG LABTECH’s Omega Series of readers, helps solve this problem.

For RT-QuIC to be successfully performed in a microplate reader, three requirements need to be met:  the assay must be incubated at 42°C for the entire measurement period from 20-60 hours, it must have alternating cycles of one minute of shaking at high speed (700 RPM) and one minute of resting, and it must be measured every 15 minutes. BMG LABTECH microplate readers fulfill all three of these requirements with the FLUOstar Omega and POLARstar Omega microplate readers having the hardware and software to run the RT-QuIC prion assay.

To learn more about this exciting new assay please see BMG LABTECH’s Application Note or the publication: Rapid End-Point Quantitation of Prion Seeding Activity with Sensitivity Comparable to Bioassays in PLoS Pathog 6(12): e1001217. 

Temperature effects on trp fluorescencec
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.