TY - JOUR T1 - Quantification of Total and Mutant Huntingtin Protein Levels in Biospecimens Using a Novel alphaLISA Assay JF - eneuro JO - eNeuro DO - 10.1523/ENEURO.0234-18.2018 SP - ENEURO.0234-18.2018 AU - Barbara Baldo AU - Muhammad Umar Sajjad AU - Rachel Y. Cheong AU - Julie Bigarreau AU - Ravi Vijayvargia AU - Catriona McLean AU - Anselme L. Perrier AU - Ihn Sik Seong AU - Glenda Halliday AU - Åsa Petersén AU - Deniz Kirik Y1 - 2018/07/16 UR - http://www.eneuro.org/content/early/2018/07/16/ENEURO.0234-18.2018.abstract N2 - 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; whilst 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 inter-assay. 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.Significance Statement The HTT lowering approaches are widely investigated as possible therapies for HD. to support these strategies, there is a high need for reproducible and sensitive assays able to quantify HTT protein. In this study we describe two robust and sensitive assays based on the AlphaLISA platform, which are able to measure either the pool of wild-type and mutant HTT, polyQ independent assay, or preferentially mutant HTT, polyQ dependent assay, in biological samples. These assays constitute a very valuable tool for HD research as they apply readily accessible antibodies and have a simple implementation. Furthermore, the HTT AlphaLISA assays are suitable for use in high throughput studies and potentially could be multiplexed to monitor simultaneously different forms of HTT. ER -