title: Development of an ELISA assay for the quantification of soluble huntingtin in human blood cells.
abstract: BACKGROUND
  Huntington's disease (HD) is a monogenic disorder caused by an aberrant expansion of CAG repeats in the huntingtin gene (HTT). Pathogenesis is associated with expression of the mutant (mHTT) protein in the CNS, with its levels most likely related to disease progression and symptom severity. Since non-invasive methods to quantify HTT in the CNS do not exist, measuring amount of soluble HTT in peripheral cells represents an important step in development of disease-modifying interventions in HD.
  
  
  RESULTS
  An ELISA assay using commercially available antibodies was developed to quantify HTT levels in complex matrices like mammalian cell cultures lysates and human samples. The immunoassay was optimized using a recombinant full-length HTT protein, and validated both on wild-type and mutant HTT species. The ability of the assay to detect significant variations of soluble HTT levels was evaluated using an HSP90 inhibitor that is known to enhance HTT degradation. Once optimized, the bioassay was applied to peripheral blood mononuclear cells (PBMCs) from HD patients, demonstrating good potential in tracking the disease course.
  
  
  CONCLUSIONS
  The method described here represents a validated, simple and rapid bio-molecular assay to evaluate soluble HTT levels in blood cells as useful tool in disease and pharmacodynamic marker identification for observational and clinical trials.
authors.affiliation: Pharmacology Department, Siena Biotech SpA, Strada del Petriccio e Belriguardo, 35, 53100 Siena, Italy. lmassai@sienabiotech.it.
authors.forename: Luisa
authors.initials: L
authors.lastname: Massai
authors.forename: Lara
authors.initials: L
authors.lastname: Petricca
authors.forename: Letizia
authors.initials: L
authors.lastname: Magnoni
authors.forename: Luca
authors.initials: L
authors.lastname: Rovetini
authors.forename: Salman
authors.initials: S
authors.lastname: Haider
authors.forename: Ralph
authors.initials: R
authors.lastname: Andre
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Tabrizi
authors.forename: Sigurd D
authors.initials: SD
authors.lastname: Süssmuth
authors.forename: Bernhard G
authors.initials: BG
authors.lastname: Landwehrmeyer
authors.forename: Andrea
authors.initials: A
authors.lastname: Caricasole
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Pollio
authors.forename: Simonetta
authors.initials: S
authors.lastname: Bernocco
mesh_terms: D000906:Antibodies / Q000276:immunology; D001774:Blood Chemical Analysis / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D057809:HEK293 Cells; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000378:metabolism / Q000473:pathology; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D013058:Mass Spectrometry; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000097:blood* / Q000235:genetics / Q000592:standards; D011786:Quality Control; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000592:standards
chemical_list: D000906:Antibodies; C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins