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Development of an ELISA assay for the quantification of soluble huntingtin in human blood cells.

Massai L, Petricca L, Magnoni L, Rovetini L, Haider S, Andre R, Tabrizi SJ, Süssmuth SD, Landwehrmeyer BG, Caricasole A, Pollio G, Bernocco S - BMC Biochem. (2013)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pharmacology Department, Siena Biotech SpA, Strada del Petriccio e Belriguardo, 35, 53100 Siena, Italy. lmassai@sienabiotech.it.

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.

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Related in: MedlinePlus

HTT detection in blood cells sample. Quantification by HTT-ELISA on human PBMC total lysates from HD patients at different disease stages, premanifest and healthy volunteers controls (HV). Gene carrier samples were significantly different from healthy controls (one-way ANOVA followed by Bonferroni test for multiple comparisons wrt HV group, p < 0.001). Individual estimates and median values are displayed in logarithmic scale.
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Figure 4: HTT detection in blood cells sample. Quantification by HTT-ELISA on human PBMC total lysates from HD patients at different disease stages, premanifest and healthy volunteers controls (HV). Gene carrier samples were significantly different from healthy controls (one-way ANOVA followed by Bonferroni test for multiple comparisons wrt HV group, p < 0.001). Individual estimates and median values are displayed in logarithmic scale.

Mentions: A bioassay designed to measure soluble HTT level multiple times over long periods of time in clinical trials requires a minimally invasive procedure to obtain suitable material for analysis. Therefore, we chose PBMCs, as they are easily obtained from a patient’s blood sample. Preliminary experiments using lysates of fresh PBMCs obtained from 6 mL of pooled rat blood allowed us to measure HTT at a concentration of 0.25 ± 0.03 nM (mean ± SD). This demonstrated that the sensitivity of the assay was sufficient to quantify native protein using 100 μL/well of total lysate without using any sample enrichment procedure. To verify the suitability of the assay for the quantification of the HTT protein in specimens similar to those commonly available in clinical trials, we analyzed lysates from frozen human PBMC pellets. The complete set of samples, five subjects of each group, included healthy volunteers, premanifest and HD patients at early, moderate and advanced disease-stage. To ensure assay reproducibility, in vitro aggregation of high molecular weight HTT protein was prevented by processing each PBMC sample as described in the Methods section. Soluble HTT levels and total protein content were subsequently measured and results were expressed for each sample as ratio between HTT quantities and total protein content (Figure 4). HTT quantification was repeated in independent experiments, loading all the samples to be compared on the same plate to avoid inter-plate effects. The outcome of these series of experiments clearly demonstrated that HD patients have significantly lower levels of soluble HTT in their PBMCs compared with healthy controls. This negative modulation is even more evident when symptoms of the disease become manifest, suggesting altered HTT processing and clearance [31,32] as the disease progresses.


Development of an ELISA assay for the quantification of soluble huntingtin in human blood cells.

Massai L, Petricca L, Magnoni L, Rovetini L, Haider S, Andre R, Tabrizi SJ, Süssmuth SD, Landwehrmeyer BG, Caricasole A, Pollio G, Bernocco S - BMC Biochem. (2013)

HTT detection in blood cells sample. Quantification by HTT-ELISA on human PBMC total lysates from HD patients at different disease stages, premanifest and healthy volunteers controls (HV). Gene carrier samples were significantly different from healthy controls (one-way ANOVA followed by Bonferroni test for multiple comparisons wrt HV group, p < 0.001). Individual estimates and median values are displayed in logarithmic scale.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221641&req=5

Figure 4: HTT detection in blood cells sample. Quantification by HTT-ELISA on human PBMC total lysates from HD patients at different disease stages, premanifest and healthy volunteers controls (HV). Gene carrier samples were significantly different from healthy controls (one-way ANOVA followed by Bonferroni test for multiple comparisons wrt HV group, p < 0.001). Individual estimates and median values are displayed in logarithmic scale.
Mentions: A bioassay designed to measure soluble HTT level multiple times over long periods of time in clinical trials requires a minimally invasive procedure to obtain suitable material for analysis. Therefore, we chose PBMCs, as they are easily obtained from a patient’s blood sample. Preliminary experiments using lysates of fresh PBMCs obtained from 6 mL of pooled rat blood allowed us to measure HTT at a concentration of 0.25 ± 0.03 nM (mean ± SD). This demonstrated that the sensitivity of the assay was sufficient to quantify native protein using 100 μL/well of total lysate without using any sample enrichment procedure. To verify the suitability of the assay for the quantification of the HTT protein in specimens similar to those commonly available in clinical trials, we analyzed lysates from frozen human PBMC pellets. The complete set of samples, five subjects of each group, included healthy volunteers, premanifest and HD patients at early, moderate and advanced disease-stage. To ensure assay reproducibility, in vitro aggregation of high molecular weight HTT protein was prevented by processing each PBMC sample as described in the Methods section. Soluble HTT levels and total protein content were subsequently measured and results were expressed for each sample as ratio between HTT quantities and total protein content (Figure 4). HTT quantification was repeated in independent experiments, loading all the samples to be compared on the same plate to avoid inter-plate effects. The outcome of these series of experiments clearly demonstrated that HD patients have significantly lower levels of soluble HTT in their PBMCs compared with healthy controls. This negative modulation is even more evident when symptoms of the disease become manifest, suggesting altered HTT processing and clearance [31,32] as the disease progresses.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pharmacology Department, Siena Biotech SpA, Strada del Petriccio e Belriguardo, 35, 53100 Siena, Italy. lmassai@sienabiotech.it.

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.

Show MeSH
Related in: MedlinePlus