Development
of an ELISA assay for the quantification of soluble huntingtin in human blood
cells
Luisa
Massai, Lara
Petricca, Letizia Magnoni, Luca Rovetini, Salman
Haider, Ralph Andre, Sarah J Tabrizi, Sigurd
D Süssmuth, Bernhard G Landwehrmeyer, Andrea Caricasole, Giuseppe
Pollio and Simonetta Bernocco
BMC
Biochemistry 2013, 14:34 doi:10.1186/1471-2091-14-34
Published:
25 November 2013
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. Tomado
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