Quantification of Total and Mutant Huntingtin Protein Levels in Biospecimens Using a Novel alphaLISA Assay.

Download
Author
Baldo, B; Sajjad, MU; Cheong, RY; Bigarreau, J; Vijayvargia, R; McLean, C; Perrier, AL; Seong, IS; Halliday, G; Petersén, Å; ...Date
2018-07Source Title
eNeuroPublisher
Society for NeuroscienceUniversity of Melbourne Author/s
McLean, CatrionaAffiliation
Florey Department of Neuroscience and Mental HealthMetadata
Show full item recordDocument Type
Journal ArticleCitations
Baldo, B., Sajjad, M. U., Cheong, R. Y., Bigarreau, J., Vijayvargia, R., McLean, C., Perrier, A. L., Seong, I. S., Halliday, G., Petersén, Å. & Kirik, D. (2018). Quantification of Total and Mutant Huntingtin Protein Levels in Biospecimens Using a Novel alphaLISA Assay.. eNeuro, 5 (4), pp.ENEURO.0234-18.2018. https://doi.org/10.1523/ENEURO.0234-18.2018.Access Status
Open AccessOpen Access at PMC
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6179573Abstract
The neurodegenerative Huntington's disease (HD) is caused by a polyglutamine (polyQ) amplification in the huntingtin protein (HTT). Currently there is no effective therapy available for HD; however, several efforts are directed to develop and optimize HTT-lowering methods to improve HD phenotypes. To validate these approaches, there is an immediate need for reliable, sensitive, and easily accessible methods to quantify HTT expression. Using the AlphaLISA platform, we developed two novel sensitive and robust assays for quantification of HTT in biological samples using commercially available antibodies. The first, a polyQ-independent assay, measures the total pool of HTT, while the second, a polyQ-dependent assay, preferentially detects the mutant form of HTT. Using purified HTT protein standards and brain homogenates from an HD mouse model, we determine a lower limit of quantification of 1 and 3 pm and optimal reproducibility with CV values lower than 7% for intra- and 20% for interassay. In addition, we used the assays to quantify HTT in neural stem cells generated from patient-derived induced pluripotent stem cells in vitro and in human brain tissue lysates. Finally, we could detect changes in HTT levels in a mouse model where mutant HTT was conditionally deleted in neural tissue, verifying the potential to monitor the outcome of HTT-lowering strategies. This analytical platform is ideal for high-throughput screens and thus has an added value for the HD community as a tool to optimize novel therapeutic approaches aimed at modulating HTT protein levels.
Export Reference in RIS Format
Endnote
- Click on "Export Reference in RIS Format" and choose "open with... Endnote".
Refworks
- Click on "Export Reference in RIS Format". Login to Refworks, go to References => Import References