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Development of an ELISA assay for the quantification of soluble huntingtin in human blood cells
Luisa Massai1, Lara Petricca, Letizia Magnoni
1Pharmacology Department, Siena Biotech SpA, Strada del Petriccio e Belriguardo, 35, 53100 Siena, Italy. lmassai@sienabiotech.it.
Insights
A new ELISA assay quantifies soluble huntingtin protein (HTT) in blood cells. This method aids in tracking Huntington's disease progression and developing new therapies.
Area of Science:
- Neuroscience
- Genetics
- Biochemistry
Background:
- Huntington's disease (HD) is a monogenic disorder caused by CAG repeat expansion in the huntingtin gene (HTT).
- Mutant huntingtin protein (mHTT) in the CNS is linked to HD progression and symptom severity.
- Lack of non-invasive CNS HTT quantification necessitates peripheral measurement for therapeutic development.
Purpose of the Study:
- To develop and validate a bio-molecular assay for quantifying soluble HTT levels in peripheral cells.
- To establish a tool for tracking HD progression and identifying pharmacodynamic markers.
Main Methods:
- Developed and optimized an ELISA assay using commercially available antibodies.
- Validated the assay on wild-type and mutant HTT species in complex matrices.
- Applied the assay to peripheral blood mononuclear cells (PBMCs) from HD patients.
Main Results:
- The ELISA assay accurately quantifies soluble HTT levels in cell lysates and human samples.
- The assay detected variations in HTT levels, validated using an HSP90 inhibitor.
- Application to HD patient PBMCs showed potential for tracking disease course.
Conclusions:
- The described method is a validated, simple, and rapid bio-assay for soluble HTT in blood cells.
- This assay can serve as a valuable tool for disease marker identification in HD observational and clinical trials.
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.
