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

Baldo, Barbara LU ; Sajjad, Muhammad Umar ; Cheong, Rachel Y. LU ; Bigarreau, Julie ; Vijayvargia, Ravi ; McLean, Catriona ; Perrier, Anselme L. ; Seong, Ihn Sik ; Halliday, Glenda and Petersén, Åsa LU , et al. (2018) In eNeuro 5(4).
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... (More)

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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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
AlphaLISA, Huntingtin, Huntington’s disease, immunoassay, polyglutamines
in
eNeuro
volume
5
issue
4
publisher
Society for Neuroscience
external identifiers
  • scopus:85054777070
  • pmid:30310861
ISSN
2373-2822
DOI
10.1523/ENEURO.0234-18.2018
language
English
LU publication?
yes
id
5f486647-462e-40a0-a610-5338158cffc8
date added to LUP
2018-10-30 08:49:22
date last changed
2024-04-01 13:55:44
@article{5f486647-462e-40a0-a610-5338158cffc8,
  abstract     = {{<p>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.</p>}},
  author       = {{Baldo, Barbara and Sajjad, Muhammad Umar and Cheong, Rachel Y. and Bigarreau, Julie and Vijayvargia, Ravi and McLean, Catriona and Perrier, Anselme L. and Seong, Ihn Sik and Halliday, Glenda and Petersén, Åsa and Kirik, Deniz}},
  issn         = {{2373-2822}},
  keywords     = {{AlphaLISA; Huntingtin; Huntington’s disease; immunoassay; polyglutamines}},
  language     = {{eng}},
  number       = {{4}},
  publisher    = {{Society for Neuroscience}},
  series       = {{eNeuro}},
  title        = {{Quantification of Total and Mutant Huntingtin Protein Levels in Biospecimens Using a Novel alphaLISA Assay}},
  url          = {{http://dx.doi.org/10.1523/ENEURO.0234-18.2018}},
  doi          = {{10.1523/ENEURO.0234-18.2018}},
  volume       = {{5}},
  year         = {{2018}},
}