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  • Florey Department of Neuroscience and Mental Health
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    Quantification of Total and Mutant Huntingtin Protein Levels in Biospecimens Using a Novel alphaLISA Assay.

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    2
    Author
    Baldo, B; Sajjad, MU; Cheong, RY; Bigarreau, J; Vijayvargia, R; McLean, C; Perrier, AL; Seong, IS; Halliday, G; Petersén, Å; ...
    Date
    2018-07
    Source Title
    eNeuro
    Publisher
    Society for Neuroscience
    University of Melbourne Author/s
    McLean, Catriona
    Affiliation
    Florey Department of Neuroscience and Mental Health
    Metadata
    Show full item record
    Document Type
    Journal Article
    Citations
    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 Access
    URI
    http://hdl.handle.net/11343/253631
    DOI
    10.1523/ENEURO.0234-18.2018
    Open Access at PMC
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6179573
    Abstract
    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.

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