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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

Full-length HTT-Q138 purification and standard curve for HTT-ELISA assay. Panel A, Gel coomassie staining of samples from the HTT-Q138 purification process (lane 1: molecular weight standard; lane 2: 30 μg RL1 cell lysate before induction; lane 3: 30 μg RL1 cell lysate 24 h post induction; lane 4: anti-FLAG affinity gel flow trough; lane 5–6: gel washes; lane 7–9: 5 μL of eluted fractions). Panel B, Western blot stained with anti-FLAG (lanes sequence as in panel A). Panel C, HTT-ELISA assay standard curve displays absorbance calibration values in duplicates individually. Curve showed is a representative example of multiple assays.
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Figure 1: Full-length HTT-Q138 purification and standard curve for HTT-ELISA assay. Panel A, Gel coomassie staining of samples from the HTT-Q138 purification process (lane 1: molecular weight standard; lane 2: 30 μg RL1 cell lysate before induction; lane 3: 30 μg RL1 cell lysate 24 h post induction; lane 4: anti-FLAG affinity gel flow trough; lane 5–6: gel washes; lane 7–9: 5 μL of eluted fractions). Panel B, Western blot stained with anti-FLAG (lanes sequence as in panel A). Panel C, HTT-ELISA assay standard curve displays absorbance calibration values in duplicates individually. Curve showed is a representative example of multiple assays.

Mentions: Recombinant human HTT full-length protein carrying a 3XFLAG tag at the N-terminus and a polyQ stretch of 138 glutamine residues (HTT-Q138) was produced using an inducible cell clone 293/T-Rex® Q138-CRE-RL1 (RL1) expressing intracellular HTT protein upon doxycycline induction [28]. To maximize yield and avoid significant degradation of the protein, induction times ranging from 12 to 96 hours were tested on small scale samples. We chose an induction time of 24 hours for the HTT-Q138 large scale preparation, since at that time HTT expression was stable. Figure 1A shows a typical result of the protein purification process. Purified HTT-Q138 showed an apparent molecular weight of ~340 kDa in agreement with the calculated value of 348 kDa. Recombinant protein was recognized by anti-FLAG antibody in cell lysate and efficiently captured by the same antibody immobilized onto the resin. Subsequently, it was eluted from the resin, by the competing FLAG-peptide; typically, we were able to obtain ~300 μg of HTT-Q138 from 1.2×109 cells with a purity of more than 90%, as evaluated by Coomassie stained NuPAGE gels. The identity of the purified protein was confirmed by Western blotting using anti-HTT specific antibodies (data not shown). Tandem mass spectrometry analysis of purified protein samples, digested with three different enzymes (trypsin, Glu-C and chymotrypsin), identified 1044 unique peptides of the protein, which corresponded to a sequence coverage of 86% and confirmed the purity of HTT-Q138.


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)

Full-length HTT-Q138 purification and standard curve for HTT-ELISA assay. Panel A, Gel coomassie staining of samples from the HTT-Q138 purification process (lane 1: molecular weight standard; lane 2: 30 μg RL1 cell lysate before induction; lane 3: 30 μg RL1 cell lysate 24 h post induction; lane 4: anti-FLAG affinity gel flow trough; lane 5–6: gel washes; lane 7–9: 5 μL of eluted fractions). Panel B, Western blot stained with anti-FLAG (lanes sequence as in panel A). Panel C, HTT-ELISA assay standard curve displays absorbance calibration values in duplicates individually. Curve showed is a representative example of multiple assays.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Full-length HTT-Q138 purification and standard curve for HTT-ELISA assay. Panel A, Gel coomassie staining of samples from the HTT-Q138 purification process (lane 1: molecular weight standard; lane 2: 30 μg RL1 cell lysate before induction; lane 3: 30 μg RL1 cell lysate 24 h post induction; lane 4: anti-FLAG affinity gel flow trough; lane 5–6: gel washes; lane 7–9: 5 μL of eluted fractions). Panel B, Western blot stained with anti-FLAG (lanes sequence as in panel A). Panel C, HTT-ELISA assay standard curve displays absorbance calibration values in duplicates individually. Curve showed is a representative example of multiple assays.
Mentions: Recombinant human HTT full-length protein carrying a 3XFLAG tag at the N-terminus and a polyQ stretch of 138 glutamine residues (HTT-Q138) was produced using an inducible cell clone 293/T-Rex® Q138-CRE-RL1 (RL1) expressing intracellular HTT protein upon doxycycline induction [28]. To maximize yield and avoid significant degradation of the protein, induction times ranging from 12 to 96 hours were tested on small scale samples. We chose an induction time of 24 hours for the HTT-Q138 large scale preparation, since at that time HTT expression was stable. Figure 1A shows a typical result of the protein purification process. Purified HTT-Q138 showed an apparent molecular weight of ~340 kDa in agreement with the calculated value of 348 kDa. Recombinant protein was recognized by anti-FLAG antibody in cell lysate and efficiently captured by the same antibody immobilized onto the resin. Subsequently, it was eluted from the resin, by the competing FLAG-peptide; typically, we were able to obtain ~300 μg of HTT-Q138 from 1.2×109 cells with a purity of more than 90%, as evaluated by Coomassie stained NuPAGE gels. The identity of the purified protein was confirmed by Western blotting using anti-HTT specific antibodies (data not shown). Tandem mass spectrometry analysis of purified protein samples, digested with three different enzymes (trypsin, Glu-C and chymotrypsin), identified 1044 unique peptides of the protein, which corresponded to a sequence coverage of 86% and confirmed the purity of HTT-Q138.

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