title: Quantification assays for total and polyglutamine-expanded huntingtin proteins.
abstract: The expansion of a CAG trinucleotide repeat in the huntingtin gene, which produces huntingtin protein with an expanded polyglutamine tract, is the cause of Huntington's disease (HD). Recent studies have reported that RNAi suppression of polyglutamine-expanded huntingtin (mutant HTT) in HD animal models can ameliorate disease phenotypes. A key requirement for such preclinical studies, as well as eventual clinical trials, aimed to reduce mutant HTT exposure is a robust method to measure HTT protein levels in select tissues. We have developed several sensitive and selective assays that measure either total human HTT or polyglutamine-expanded human HTT proteins on the electrochemiluminescence Meso Scale Discovery detection platform with an increased dynamic range over other methods. In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions. We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples. Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples.
authors.affiliation: CHDI Management/CHDI Foundation, Los Angeles, California, United States of America.
authors.forename: Douglas
authors.initials: D
authors.lastname: Macdonald
authors.affiliation: Galapagos B.V., Leiden, The Netherlands.
authors.forename: Michela A
authors.initials: MA
authors.lastname: Tessari
authors.affiliation: BioFocus, a Charles River company, Leiden, The Netherlands.
authors.forename: Ivette
authors.initials: I
authors.lastname: Boogaard
authors.affiliation: BioFocus, a Charles River company, Saffron Walden, United Kingdom.
authors.forename: Melanie
authors.initials: M
authors.lastname: Smith
authors.affiliation: Galapagos B.V., Leiden, The Netherlands.
authors.forename: Kristiina
authors.initials: K
authors.lastname: Pulli
authors.affiliation: BioFocus, a Charles River company, Leiden, The Netherlands.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Szynol
authors.affiliation: BioFocus, a Charles River company, Leiden, The Netherlands.
authors.forename: Faywell
authors.initials: F
authors.lastname: Albertus
authors.affiliation: BioFocus, a Charles River company, Saffron Walden, United Kingdom.
authors.forename: Marieke B A C
authors.initials: MB
authors.lastname: Lamers
authors.affiliation: BioFocus, a Charles River company, Leiden, The Netherlands.
authors.forename: Sipke
authors.initials: S
authors.lastname: Dijkstra
authors.affiliation: Evotec AG, Hamburg, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kordt
authors.affiliation: Evotec AG, Hamburg, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Reindl
authors.affiliation: Evotec AG, Hamburg, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Herrmann
authors.affiliation: BioFocus, a Charles River company, Saffron Walden, United Kingdom.
authors.forename: George
authors.initials: G
authors.lastname: McAllister
authors.affiliation: BioFocus, a Charles River company, Leiden, The Netherlands.
authors.forename: David F
authors.initials: DF
authors.lastname: Fischer
authors.affiliation: CHDI Management/CHDI Foundation, Los Angeles, California, United States of America.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Munoz-Sanjuan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001681:Biological Assay / Q000379:methods*; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D005260:Female; D006801:Humans; D000071058:Huntingtin Protein; D008163:Luminescent Measurements; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D051381:Rats; D012995:Solubility
chemical_list: D000906:Antibodies; C086055:HTT protein, human; C506812:Htt protein, mouse; C499524:Htt protein, rat; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010455:Peptides; C097188:polyglutamine