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Prion diseases, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis (ALS) are all part of the landscape of neurodegenerative diseases linked to protein misfolding. Each disease involves its own unique misfolded protein or combination of proteins, protein deposits, and accumulations of amyloid fibers leading to specific progressive neuronal pathological features and clinical symptoms.

Microplate readers are used widely to study protein misfolding. For this purpose, multiple applications such as enzyme-linked immunosorbent assays (ELISAs), cell-based assays, and protein stability and protein-protein interaction assays were developed over the years.

Measuring protein misfolding and aggregation on microplate readers

One popular example for the study of the aggregation of misfolded proteins, the real-time quaking-induced conversion (RT-QuIC) assay, was developed as a rapid and sensitive diagnostic tool for prion detection. RT-QuIC is a seeding assay that uses a cell-free conversion reaction in which recombinant noninfectious PrPC prion proteins are converted by an original seed into the PrPSc pathogenic and infectious isoform, and in which aggregation and exponential amplification enables detection of otherwise undetectable prion levels (Figure 1). RT-QuIC assays use fluorescence intensity detection of thioflavin T and cycled shaking to produce prion protein multimers when a PrPSc prion seed is present (See AN 232).

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Figure 1. Example of prion protein aggregation data generated by RT-QuIC.

The RT-QuIC prion assay relies on a microplate reader for signal quantification, as well as for shaking and temperature conditions conducive for seeding and formation of aggregates. BMG LABTECH microplate readers play a central role in prion research because the RT-QuIC assay was originally developed on the FLUOstar® Omega, the reader that has become the worldwide standard for prion detection for a variety of species, including human prions. The RT-QuIC prion assay takes advantage of the solid construction of BMG LABTECH microplate readers to perform on and off cycles of shaking for an extended time (up to 60 hours), while periodically reading the plate to monitor changes in fluorescent signal as thioflavin T is incorporated into prion aggregates.

As an alternative to thioflavin T–based aggregation assays, prefibrillar species of amyloidogenic proteins can be monitored by fluorescence polarization using the fluorescent probe TPE-TPP, as described in the application note “Novel aggregation-specific fluorogen monitors prefibrillar protein aggregation by fluorescence polarisation.”

Future directions and challenges in protein misfolding research

New drugs are actively being sought for the treatment of neurodegenerative diseases related to protein misfolding and aggregation. In clinical trials, there is a big need for translatable biomarkers that track the progression and severity of the neurodegenerative diseases impacted by protein misfolding. Some clinical trials have recently made progress for targeting beta-amyloid in Alzheimer’s disease, but much work lies ahead. Progress in artificial intelligence and modeling techniques have made significant contributions to the study of protein folding and misfolding which should bring further advances to the drug discovery space.

BMG LABTECH offers a wide range of single-mode to multimode microplate readers that are ideally suited for studying protein misfolding. While the FLUOstar Omega remains the gold standard for RT-QuIC and seeding assays, the PHERAstar® FSX was specifically conceived for screening campaigns and is your go-to reader for high-performance high-throughput screening. Both the VANTAstar® and CLARIOstar® Plus allow for wavelength flexibility and include Enhanced Dynamic Range technology for superior performance and user-friendliness. Collectively, these multimode readers combine high performance with miniaturized assays and short measurement times, and they offer considerable savings on materials and other resources.


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If you are interested in further details about microplate-based evaluation of protein misfolding, please contact us at [email protected]

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