F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.153670.2 Data Note Articles A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence

[version 2; peer review: 5 approved, 1 approved with reservations]

 Fanti Rebeka Investigation Validation Writing – Original Draft Preparation 1 Ayoubi Riham Data Curation Investigation Methodology Validation Visualization Writing – Original Draft Preparation 2 Alende Charles Investigation Visualization https://orcid.org/0009-0005-4611-6134 2 Fotouhi Maryam Investigation 2 González Bolívar Sara Investigation https://orcid.org/0000-0002-4299-8281 2 Chandrasekaran Renu Investigation 1 Southern Kathleen Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-4125-3608 2 Edwards Aled M. Funding Acquisition Supervision 1 Harding Rachel J. Supervision Validation https://orcid.org/0000-0002-1134-391X 1 3 Laflamme Carl Funding Acquisition Methodology Project Administration Resources Supervision https://orcid.org/0000-0001-5906-025X a 2 NeuroSGC/YCharOS/EDDU collaborative group ABIF consortium Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, Québec, Canada Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, Canada carl.laflamme@mcgill.ca

Competing interests: For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: Abcam, Aviva Systems Biology, Bio-Techne, Cell Signalling Technology, Developmental Studies Hybridoma Bank, GeneTex, Horizon Discovery, Proteintech, Synaptic Systems, Thermo Fisher Scientific.

 2 1 2025 2024 13 922 23 12 2024 Copyright: © 2025 Fanti R et al. 2025 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Huntingtin encodes a 3144 amino acid protein, with a polyglutamine repeat tract at the N-terminus. Expansion of this repeat tract above a pathogenic threshold of 36 repeats is the causative mutation of Huntington's disease, a neurodegenerative disorder characterized by loss of striatal neurons. Here we have characterized twenty Huntingtin commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

 UniProt ID P42858 HTT Huntingtin antibody characterization antibody validation western blot immunoprecipitation immunofluorescence Ontario Genomics OGI-210 Genome Quebec Mitacs Genome Canada This work was supported by a grant from the Government of Canada through Genome Canada, Genome Quebec, and Ontario Genomics (grant no. OGI-210). RF and RA are supported by Mitacs fellowships. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Amendments from Version 1

An error occurred as only half of the antibodies tested by our team were included in Figure 3. The original submission featured a split Figure 3 with Parts 1 and 2 uploaded separately. The revised Figure 3 now includes all 20 HTT antibodies tested.

 Introduction

Huntington’s Disease (HD) is a neurodegenerative disorder inherited in an autosomal dominant manner, presenting with a spectrum of progressive motor, cognitive, and psychological impairments, typically with adult-onset of symptoms. 1 Although the HD causative gene, HTT, was discovered over three decades ago, there are still no disease-modifying treatments available for patients, and progress unpicking the molecular pathology of the disease remains slow. 2

HD arises from a heterozygous expansion mutation of the trinucleotide CAG repeat tract in exon 1 of HTT, located on chromosome 4, above a critical threshold of ~36 repeats. This mutation results in expansion of the polyglutamine stretch at the N-terminus of the 3144 amino acid Huntingtin protein. Huntingtin functions as a scaffold protein, engaging in extensive protein-protein interactions, 3 forming various multi-protein complexes to carry-out its diverse array of functions. Modulation of this interaction network by the polyglutamine expansion contributes to degeneration within the central nervous system, affecting medium spiny neurons at the onset of disease. 4 , 5

The low expression level, complex interactome and large size of the 348 kDa Huntingtin protein have given rise to technical challenges which have hindered precise determination of its molecular function, or how this is altered in disease. In particular, the use of different Huntingtin antibodies by scientists in the HD research community, often mapping to structurally distant epitopes, can yield different or even conflicting results, further conflating interrogation of this protein. 6 – 8

This research is part of a broader collaborative initiative in which academics, funders and commercial antibody manufacturers are working together to address antibody reproducibility issues by characterizing commercial antibodies for human proteins using standardized protocols, and openly sharing the data. 9 – 11 Here we evaluated the performance of twenty commercial antibodies for Huntingtin for use in western blot, immunoprecipitation, and immunofluorescence, enabling biochemical and cellular assessment of Huntingtin properties and function. The platform for antibody characterization used to carry out this study was endorsed by a committee of industry and academic representatives. It consists of identifying human cell lines with adequate target protein expression and the development/contribution of equivalent knockout (KO) cell lines, followed by antibody characterization procedures using most commercially available antibodies against the corresponding target protein. The standardized consensus antibody characterization protocols are openly available on Protocol Exchange (DOI: 10.21203/rs.3.pex-2607/v1). 12

The authors do not engage in result analysis or offer explicit antibody recommendations. A limitation of this study is the use of universal protocols - any conclusions remain relevant within the confines of the experimental setup and cell line used in this study. Our primary aim is to deliver top-tier data to the scientific community, grounded in Open Science principles. This empowers experts to interpret the characterization data independently, enabling them to make informed choices regarding the most suitable antibodies for their specific experimental needs. Guidelines on how to interpret antibody characterization data found in this study are featured on the YCharOS gateway. 13

Results and discussion

Our standard protocol involves comparing readouts from WT (wild type) and KO cells. 14 , 15 The first step is to identify a cell line(s) that expresses sufficient levels of a given protein to generate a measurable signal. To this end, we examined the DepMap transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log 2 (transcripts per million “TPM” + 1), which we have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655). We generated an HTT KO line in DMS 53 as it expresses the endogenous HTT transcript at 6.1 log 2 (TPM+1), which is above the average range of cancer cell lines analyzed. A commercial HAP1 HTT KO is also available; HAP1 expresses HTT at 3.7 log 2 (TPM+1) RNA level. A HEK293T HTT KO cell line has been developed and used elsewhere 16 ( Table 1). All three cell line backgrounds were evaluated by western blot using a high-performing Huntingtin antibody detected in Figure 1. DMS 53 was identified as the most suitable cell line ( Figure 2), which can be explained by its high expression of the HTT transcript compared to other two cell lines. Thus, DMS 53 WT and KO cell lines were generated and used to evaluate the antibodies in all applications.

Summary of the cell lines used.
Institution Catalog number RRID (Cellosaurus) Cell line Genotype
ATCC CRL-2062 CVCL_1177 DMS 53 WT
Academic non-commercial CVCL_D6U0 DMS 53 HTT KO
ATCC CRL-3216 CVCL_0063 HEK 293T WT
Academic non-commercial CVCL_D7EP HEK 293T HTT KO 16
Horizon Discovery C631 CVCL_Y019 HAP1 WT
Horizon Discovery HZGHC004595c006 CVCL_SR86 HAP1 HTT KO
Huntingtin antibody screening by western blot.

Lysates of DMS 53 (WT and HTT KO) were prepared and 30 μg of protein were processed for western blot with the indicated Huntingtin antibodies. Tris-Glycine 4-20% gels were used for SDS-PAGE. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given for antibodies ab109115**, ab45169**, and MA5-41256** which were titrated as the signal was too weak when following the supplier’s recommendations. Antibody dilution used: ab109115** at 1/2000, ab45169** at 1/5000, ABCD_AG854** at 1/10, ABCD_AG855** at 1/10, A19064** at 1/1000, 5656** at 1/1000, CH03023* at 1/1000, MW1-S* at 1/10, MW3-S* at 1/10, MW4-S* at 1/10, MW5-S* at 1/10, MW6-S* at 1/10, MW7-S* at 1/10, MW8-S* at 1/10, GTX132433 at 1/500, GTX638832** at 1/500, 710695** at 1/200, MA3-040* at 1/1000, MA5-16703* at 1/500, MA5-41256** at 1/2000. Predicted band size: 347 kDa. *Monoclonal antibody, **Recombinant antibody.

Note: MW1-S*, MW3-S*, MW4-S*, MW5-S*, MW6-S*, MW7-S* and MW8-S* are expected to only recognize an altered conformation of the polyQ domain generated as the polyQ domain of Huntingtin increases in length.

 Huntingtin western blot on various cell lysates.

Lysates of WT and HTT KO in DMS 53, HEK 293T and HAP1 were prepared, and 30 μg of protein was processed for western blot with the indicated Huntingtin antibodies ab45169** at 1/5000 and GTX638832** at 1/500. Tris-Glycine 4-20% gels were used for SDS-PAGE. The Ponceau stained transfer is shown as a loading control. Predicted band size: 347 kDa. **Recombinant antibody.

For western blot experiments, WT and HTT KO protein lysates were ran on SDS-PAGE, transferred onto nitrocellulose membranes, and then probed with twenty Huntingtin antibodies in parallel ( Table 2, Figure 1).

Summary of the Huntingtin antibodies tested.
Company Catalog number Lot number RRID (Antibody Registry) Clonality Clone ID Host Concentration (μg/μl) Vendors recommended applications
Abcam ab109115 ** 1003147-4 AB_10863082 recombinant-mono EPR5526 rabbit 1.52 Wb, IF
Abcam ab45169 ** 1022500 AB_733062 recombinant-mono EP867Y rabbit 1.55 Wb, IF
ABCD Antibodies ABCD_AG854 ** 10/27/2023 AB_3076339 recombinant-mono 12.3 rabbit 0.004 n/a
ABCD Antibodies ABCD_AG855 ** 10/27/2023 AB_3076340 recombinant-mono C4 rabbit 0.19 n/a
ABclonal A19064 ** 4000000431 AB_2862557 recombinant-mono ARC0431 rabbit 0.63 Wb, IF
Cell Signaling Technology 5656 ** 6 AB_10827977 recombinant-mono D7F7 rabbit 0.10 Wb, IF
Coriell Institute CH03023 * 03.17.21 AB_3096092 monoclonal 2B7 mouse 1.71 n/a
DSHB MW1-S * 44322 AB_528290 monoclonal MW1-S mouse 0.02 Wb, IP, IF
DSHB MW3-S * 43216 AB_528292 monoclonal MW3-S mouse 0.01 Wb, IF
DSHB MW4-S * 43251 AB_528293 monoclonal MW4-S mouse 0.02 Wb, IF
DSHB MW5-S * 44742 AB_528294 monoclonal MW5-S mouse 0.03 Wb, IF
DSHB MW6-S * 43230 AB_528295 monoclonal MW6-S mouse 0.06 Wb, IF
DSHB MW7-S * 43461 AB_528296 monoclonal MW7-S mouse 0.03 Wb, IF
DSHB MW8-S * 44315 AB_528297 monoclonal MW8-S mouse 0.04 Wb, IP, IF
GeneTex GTX132433 42312 AB_2886646 polyclonal MW3-S rabbit 1.40 Wb, IF
GeneTex GTX638832 ** 45096 AB_3094813 recombinant-mono HL2483 rabbit 0.98 Wb
Thermo Fisher Scientific 710695 ** RF236710 AB_2608784 recombinant-poly 3HCLC rabbit 0.50 IF
Thermo Fisher Scientific MA3-040 * YG376237 AB_2608783 monoclonal 1HU-4C8 mouse n/a Wb, IF
Thermo Fisher Scientific MA5-16703 * YE3913821A AB_2538195 monoclonal HDB4E10 mouse 1.00 Wb, IP, IF
Thermo Fisher Scientific MA5-41256 ** YE3913382B AB_2899009 recombinant-mono JB89-34 rabbit 1.00 Wb, IF

Wb=western blot; IF=immunofluorescence; IP=immunoprecipitation; n/a=not available, DSHB=Developmental Studies Hybridoma Bank.

Monoclonal antibody,

Recombinant antibody.

We then assessed the capability of all twenty antibodies to capture Huntingtin from DMS 53 protein extracts using immunoprecipitation techniques, followed by western blot analysis. For the immunoblot step, a specific Huntingtin antibody identified previously (refer to Figure 1) was selected. Equal amounts of the starting material (SM), the unbound fraction (UB), as well as the whole immunoprecipitate (IP) eluates were separated by SDS-PAGE ( Figure 3).

Huntingtin antibody screening by immunoprecipitation.

DMS 53 lysates were prepared, and immunoprecipitation was performed using 2.0 μg of the indicated Huntingtin antibodies pre-coupled to Dynabeads protein A or protein G. The concentration of MA3-040* is unknown and therefore 5 μL of this antibody was tested. All samples were washed and processed for western blot with the indicated Huntingtin antibody. Tris-Glycine 4-20% gels were used for SDS-PAGE. For western blot, ab45169** was used at 1/5000. The Ponceau stained transfers of each blot are shown. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate. *Monoclonal antibody, **Recombinant antibody.

For immunofluorescence, twenty antibodies were screened using a mosaic strategy. First, DMS 53 WT and HTT KO cells were labelled with different fluorescent dyes in order to distinguish the two cell lines, and the Huntingtin antibodies were evaluated. Both WT and KO lines were imaged in the same field of view to reduce staining, imaging and image analysis bias ( Figure 4). Quantification of immunofluorescence intensity in hundreds of WT and KO cells was performed for each antibody tested, 12 and the images presented in Figure 3 are representative of this analysis.

Huntingtin antibody screening by immunofluorescence.

DMS 53 WT and HTT KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio on coverslips. Cells were stained with the indicated Huntingtin antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (WT), red (antibody staining) and far-red (KO) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined with green and magenta dashed line, respectively. When an antibody was recommended for immunofluorescence by the supplier, we tested it at the recommended dilution and at 1 μg/ml. The rest of the antibodies were tested at 1 and 2 μg/ml. The final concentration of each antibody was selected based on the detection range of the microscope used and a quantitative analysis not shown here. Antibody dilutions corresponding to the images shown are: ab109115** at 1/1500, ab45169** at 1/1500, ABCD_AG854** at 1/500, ABCD_AG855** at 1/200, A19064** at 1/600, 5656** at 1/100, CH03023* at 1/1700, MW1-S* at 1/20, MW3-S* at 1/10, MW4-S* at 1/10, MW5-S* at 1/15, MW6-S* at 1/60, MW7-S* at 1/15, MW8-S* at 1/20, GTX132433 at 1/500, GTX638832** at 1/500, 710695** at 1/500, MA3-040* at 1/1000, MA5-16703* at 1/1000, MA5-41256** at 1/1000. Bars = 10 μm. *Monoclonal antibody, **Recombinant antibody.

In conclusion, we have screened twenty commercial Huntingtin antibodies by western blot, immunoprecipitation, and immunofluorescence by comparing the signal produced by the antibodies in human DMS 53 WT and HTT KO cells. Several high-quality and renewable Huntingtin antibodies were identified in all applications. Researchers who wish to study Huntingtin in a different species are encouraged to select high-quality antibodies, based on the results of this study, and investigate the predicted species reactivity of the manufacturer before extending their research.

The underlying data for this study can be found on Zenodo, an open-access repository for which YCharOS has its own collection of antibody characterization reports. 17

Methods

The standardized protocols used to carry out this KO cell line-based antibody characterization platform was established and approved by a collaborative group of academics, industry researchers and antibody manufacturers. The detailed materials and step-by-step protocols used to characterize antibodies in western blot, immunoprecipitation and immunofluorescence are openly available on Protocol Exchange, a preprint server (DOI: 10.21203/rs.3.pex-2607/v1). 12

Antibodies and cell lines used

Cell lines used and primary antibodies tested in this study are listed in Tables 1 and 2, respectively. To ensure that the cell lines and antibodies are cited properly and can be easily identified, we have included their corresponding Research Resource Identifiers, or RRID. 18 , 19

Data availability Underlying data

Zenodo: Dataset for the Huntingtin antibody screening study, doi.org/10.5281/zenodo.11639052. 17

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

 Acknowledgment

We would like to thank the NeuroSGC/YCharOS/EDDU collaborative group for their important contribution to the creation of an open scientific ecosystem of antibody manufacturers and KO cell line suppliers, for the development of community-agreed protocols, and for their shared ideas, resources, and collaboration. Members of the group can be found below. We would also like to thank the Advanced BioImaging Facility (ABIF) consortium for their image analysis pipeline development and conduction (RRID:SCR_017697). Members of each group can be found below.

NeuroSGC/YCharOS collaborative group: Thomas M. Durcan, Aled M. Edwards, Peter S. McPherson, Chetan Raina and Wolfgang Reintsch.

ABIF consortium: Claire M. Brown and Joel Ryan.

Thank you to the Structural Genomics Consortium, a registered charity (no. 1097737), for your support on this project. The Structural Genomics Consortium receives funding from Bayer AG, Boehringer Ingelheim, Bristol-Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute (grant no. OGI-196), the EU and EFPIA through the Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant no. 875510), Janssen, Merck KGaA (also known as EMD in Canada and the United States), Pfizer and Takeda.

An earlier version of this of this article can be found on Zenodo (DOI: 10.5281/zenodo.11582780).

 References Ross CA Tabrizi SJ : Huntington's disease: from molecular pathogenesis to clinical treatment. Lancet Neurol. 2011;10(1):83–98. 10.1016/S1474-4422(10)70245-3 A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The Huntington's Disease Collaborative Research Group. Cell. 1993;72(6):971–983. 8458085 10.1016/0092-8674(93)90585-E Greco TM Secker C Ramos ES : Dynamics of huntingtin protein interactions in the striatum identifies candidate modifiers of Huntington disease. Cell Syst. 2022;13(4):304–320.e5. 35148841 10.1016/j.cels.2022.01.005 PMC9317655 Matlik K Baffuto M Kus L : Cell-type-specific CAG repeat expansions and toxicity of mutant Huntingtin in human striatum and cerebellum. Nat. Genet. 2024;56(3):383–394. 38291334 10.1038/s41588-024-01653-6 PMC10937393 Pressl C Matlik K Kus L : Selective vulnerability of layer 5a corticostriatal neurons in Huntington's disease. Neuron. 2024;112(6):924–941.e10. 38237588 10.1016/j.neuron.2023.12.009 Strong TV Tagle DA Valdes JM : Widespread expression of the human and rat Huntington's disease gene in brain and nonneural tissues. Nat. Genet. 1993;5(3):259–265. 8275091 10.1038/ng1193-259 Greco TM Secker C Ramos ES : Dynamics of huntingtin protein interactions in the striatum identifies candidate modifiers of Huntington disease. Cell Syst. 2022;13(4):304–20.e5. 35148841 10.1016/j.cels.2022.01.005 PMC9317655 Wanker EE Ast A Schindler F : The pathobiology of perturbed mutant huntingtin protein-protein interactions in Huntington's disease. J. Neurochem. 2019;151(4):507–519. 31418858 10.1111/jnc.14853 Ayoubi R Ryan J Biddle MS : Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications. elife. 2023;12:12. 10.7554/eLife.91645.2 Carter AJ Kraemer O Zwick M : Target 2035: probing the human proteome. Drug Discov. Today. 2019;24(11):2111–2115. 31278990 10.1016/j.drudis.2019.06.020 Licciardello MP Workman P : The era of high-quality chemical probes. Rsc Med. Chem. 2022;13(12):1446–1459. 36545432 10.1039/D2MD00291D PMC9749956 Ayoubi R Ryan J Bolivar SG : A consensus platform for antibody characterization (Version 1). Protocol. Exchange. 2024. 10.21203/rs.3.pex-2607/v1 Biddle MS Virk HS : YCharOS open antibody characterisation data: Lessons learned and progress made. F1000Res. 2023;12(12):1344. 10.12688/f1000research.141719.1 Laflamme C McKeever PM Kumar R : Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72. elife. 2019;8:8. 10.7554/eLife.48363 Alshafie W Fotouhi M Shlaifer I : Identification of highly specific antibodies for Serine/threonine-protein kinase TBK1 for use in immunoblot, immunoprecipitation and immunofluorescence. F1000Res. 2022;11:977. 10.12688/f1000research.124632.1 Jung R Lee Y Barker D : Mutations causing Lopes-Maciel-Rodan syndrome are huntingtin hypomorphs. Hum. Mol. Genet. 2021;30(3-4):135–148. 33432339 10.1093/hmg/ddaa283 PMC8248964 Ayoubi R Laflamme C : Dataset for the Huntingtin antibody screening study.[Data set]. Zenodo. 2024. Bandrowski A Pairish M Eckmann P : The Antibody Registry: ten years of registering antibodies. Nucleic Acids Res. 2023;51(D1):D358–D367. 36370112 10.1093/nar/gkac927 PMC9825422 Bairoch A : The Cellosaurus, a Cell-Line Knowledge Resource. J. Biomol. Tech. 2018;29(2):25–38. 29805321 10.7171/jbt.18-2902-002 PMC5945021 10.5256/f1000research.176211.r360581 Reviewer response for version 2 Bertoglio Daniele 1 Referee https://orcid.org/0000-0003-4205-5432 University of Antwerp, Antwerp, Belgium

Competing interests: No competing interests were disclosed.

 12 2 2025 Copyright: © 2025 Bertoglio D 2025 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

This manuscript by Fanti et al. presents a comprehensive evaluation of twenty commercially available HTT antibodies utilizing standardized experimental protocols. The study systematically assesses the suitability and sensitivity of these antibodies in Western blot, immunoprecipitation, and immunofluorescence applications, employing both wild-type and knock-out HTT cell lines. It would have been of additional support inclusion of immunohistochemistry, but this does not restrict the value of the work.

The study addresses a critical technical challenge in Huntington’s Disease (HD) research, namely the variability in antibody performance, which has posed a significant issue within the field.

The experimental approach is well conceived, designed, and executed. The data are presented in a clear and accessible manner, facilitating ease of interpretation. The findings provide valuable guidance for researchers seeking to source and utilize specific HTT antibodies in their investigations. Overall, this study constitutes a significant contribution to the HD research community, offering essential data to improve experimental consistency and reproducibility

Are sufficient details of methods and materials provided to allow replication by others?

Yes

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

In vivo and post-mortem imaging of neurodegenetive disorders, including HD

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

 10.5256/f1000research.176211.r358661 Reviewer response for version 2 Subramaniam Srinivasa 1 Referee The Scripps Research Institute, Jupiter, USA

Competing interests: No competing interests were disclosed.

 6 2 2025 Copyright: © 2025 Subramaniam S 2025 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

Fanti et al. evaluated twenty commercial antibodies against huntingtin for Western blot, immunoprecipitation, and immunofluorescence by employing a defined experimental approach that involved comparing results in deletion cell lines with isogenic parental controls. This study is a crucial guide that will significantly enhance research on huntingtin biology and Huntington's disease.

The authors employed twenty commercial antibodies and three distinct cell lines, validating the antibodies across several assays. I have a question concerning IP.

It is unclear whether the authors employed control beads as an extra control measure during the immunoprecipitation process. Figure 3 necessitates either control beads or immunoprecipitation conducted in knockout cells.

Are sufficient details of methods and materials provided to allow replication by others?

Yes

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Mechanisms of neurodegenerative disease

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

 10.5256/f1000research.176211.r360585 Reviewer response for version 2 Saiyin Hexige 1 Referee https://orcid.org/0000-0003-2993-6817 Fudan University, Shanghai, China

Competing interests: No competing interests were disclosed.

 6 2 2025 Copyright: © 2025 Saiyin H 2025 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

This well-designed study validates twenty HTT antibodies by WB and IF. Based on the reviewer's experience and other studies, HTT is unstable after fixation in human tissue and cells (Ferrante, Gutekunst et al. 1997)(ref 2). Thus, the reviewer suggests providing the details of the fixation in IF and processing the WB sample in WB, especially the staining/blotting time after the fixation or harvesting WB (such as the staining performed immediately after fixation or 48 hours after fixation et al.). 

Minor concerns:

The resolution of IF images is low. The cellular distribution patterns of HTT in cells are difficult to read. Thus, the reviewer encourages showing high-resolution images in IF, which can provide more detail of cellular distribution patterns.

HTTs are easy to aggregate (DiFiglia, Sapp et al. 1997, Gutekunst, Li et al. 1999)(ref 1 and 3). The author only detected the single HTT but not the aggregate. Does the antibody that recognizes the single HTT  also equally detect the aggregate of cells or tissue?  Does the author try to detect the aggregate by those antibodies?

Are sufficient details of methods and materials provided to allow replication by others?

Partly

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Partly

Reviewer Expertise:

Pathogenesis of HD and 3D histology and pathology of human disease.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

References : Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science .1997;277(5334) : 10.1126/science.277.5334.1990 1990-3 9302293 10.1126/science.277.5334.1990 : Heterogeneous topographic and cellular distribution of huntingtin expression in the normal human neostriatum. J Neurosci .1997;17(9) : 10.1523/JNEUROSCI.17-09-03052.1997 3052-63 9096140 10.1523/JNEUROSCI.17-09-03052.1997 : Nuclear and neuropil aggregates in Huntington's disease: relationship to neuropathology. J Neurosci .1999;19(7) : 10.1523/JNEUROSCI.19-07-02522.1999 2522-34 10087066 10.1523/JNEUROSCI.19-07-02522.1999 10.5256/f1000research.176211.r360584 Reviewer response for version 2 Landles Christian 1 Referee https://orcid.org/0000-0001-9715-6447 University College London, London, UK

Competing interests: No competing interests were disclosed.

 5 2 2025 Copyright: © 2025 Landles C 2025 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

This manuscript by Fanti et al. has comprehensively characterized twenty commercially available HTT antibodies using cell lines which were either wild-type or knock-out for the HTT gene. HTT antibody suitability and sensitivity was assessed using standardized experimental protocols using the applications of western blot, immunoprecipitation, and immunofluorescence. This experimental approach was rationally conceived and executed, and the experimental dataset has been presented in a clear manner which was easy to follow and interpret. Overall, this data will be of great use should anyone within the Huntington’s disease research community wish to source and try any specific HTT antibody for their own research needs.

Minor Comments and Recommendations

Introduction:

The expression level of the full-length HTT protein is not particularly “low” in brain tissues, it is peripheral tissues which express full-length HTT and low levels.

Results and discussion:

Table 1, it would be useful to report what lineage these cell lines used are from, and also state the CAG repeat size of these cell lines.

The MW1 to MW7 antibodies, originally characterized in Ko, et al., Brain Res. Bull., 2001, predominantly recognise mutant HTT isoforms where the pathogenic CAG tract has been expanded. Therefore, although stated in the figure legend of Fig. 1 “MW1-S*, MW3-S*, MW4-S*, MW5-S*, MW6-S*, MW7-S* and MW8-S* are expected to only recognize an altered conformation of the polyQ domain generated as the polyQ domain of Huntingtin increases in length.”, I am not sure why these antibodies were being tested here on wild-type DMS 53 cell lines where there was no expanded CAG mutation. A negative signal here was always to be expected.

The MW8 antibody, originally characterized in Ko, et al., Brain Res. Bull., 2001 [ref 1], and later mapped by Landles et al., JBC, 2010 [ref 2], has been demonstrated to be a neo-epitope antibody specifically recognizing the highly pathogenic mutant HTT1a protein isoform. It has subsequently been shown in numerous research papers to not recognise the full-length HTT protein isoform, and so likewise, a negative signal here on wild-type DMS 53 cells was always to be expected. (It would be helpful if these two points were explained fully in the main body of text and not buried in the figure legend of Fig. 1).

Since Fanti et al. was comprehensively characterizing commercial HTT antibodies, I was very surprised that the commercial monoclonal HTT-MAB5490 and HTT-MAB2166 antibodies were used in their analyses. These two monoclonal HTT antibodies are widely used throughout the entire Huntington’s disease research community, and characterising these here (maybe instead of the MW1-MW8 series) would have been more appropriate / valuable.

 Methods:

In the interest of having all the methodologies used in this already concise manuscript, it would be helpful to the reader to have all the experimental methodologies within this section, as opposed to viewing them from another link online.

Are sufficient details of methods and materials provided to allow replication by others?

Partly

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

The molecular pathogenesis of Huntingtin's disease, pre-clinical Huntington's disease mouse models, HTT protein bioassays, Molecular biology, HTT protein aggregation.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

References : New anti-huntingtin monoclonal antibodies: implications for huntingtin conformation and its binding proteins. Brain Res Bull .56(3-4) : 10.1016/s0361-9230(01)00599-8 319-29 11719267 10.1016/s0361-9230(01)00599-8 : Proteolysis of mutant huntingtin produces an exon 1 fragment that accumulates as an aggregated protein in neuronal nuclei in Huntington disease. J Biol Chem .2010;285(12) : 10.1074/jbc.M109.075028 8808-23 20086007 10.1074/jbc.M109.075028 10.5256/f1000research.168592.r315013 Reviewer response for version 1 Bowman Karen 1 Referee https://orcid.org/0009-0002-9288-7106 University of Leicester, Leicester, England, UK

Competing interests: No competing interests were disclosed.

 9 9 2024 Copyright: © 2024 Bowman K 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

The article pointed out the current technical difficulties in studying Huntington’s Disease and identified the differences in antibodies to the protein Huntingtin, used throughout the HD research community, as a particular problem. The study formed part of the wider YCharOS collaboration to characterise commercial antibodies for human proteins using standardized protocols with the intention of improving antibody reproducibility issues. The study was an evaluation of twenty commercial antibodies for the protein Huntingtin for use in western blot, immunoprecipitation, and immunofluorescence.

The study was well designed and the results were to a high technical standard and quality. The data is presented clearly and looks robust.

It was reassuring to see that a committee of industry and academic representatives had endorsed the platform used. The platform consisted of identification of human cell lines suitable for antibody characterisation studies i.e. with adequate levels of Huntingtin, followed by the development of and contribution of isogenic knockout control cell lines, which were validated at the protein level. The final step was a series of antibody characterisation procedures, limited to the most common research uses of antibodies. Limitations of the study are clearly stated.

Two minor errors were spotted. There is an inconsistency between the text and figure 3, where they mention IP was done on twenty antibodies in the text, but results for only ten antibodies are presented in the figure. Perhaps some additional text on why only ten were presented would be of use, or amend the text to reflect what is presented in the figure. A second error is that in the final results paragraph on immunofluorescence, figure 3 is cited, and linked back to, instead of figure 4.

Overall, this will be a highly useful resource for scientists in the HD research community.

Are sufficient details of methods and materials provided to allow replication by others?

Yes

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

drug discovery and diagnostics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

 10.5256/f1000research.168592.r315014 Reviewer response for version 1 Shao Jieya 1 Referee https://orcid.org/0000-0003-1859-9384 Washington University in St Louis, St. Louis, Missouri, USA

Competing interests: No competing interests were disclosed.

 28 8 2024 Copyright: © 2024 Shao J 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

In this study, the authors performed comprehensive analysis of twenty commercially available HTT antibodies for their suitability and performance in Western blot, immunoprecipitation, and immunofluorescence staining applications. The experimental approaches were logically and thoroughly designed. Data are of high-quality and clearly presented. The results will be very helpful for researchers in the HTT field. However, there seems to be two small errors.

First, in Figure 3, only the IP data of 10 antibodies were shown while in the text it was stated that 20 antibodies were analyzed.

Second, on Page 5, in the last sentence of the second paragraph, the authors seem to cite Figure 3 by mistake.

Are sufficient details of methods and materials provided to allow replication by others?

Yes

Is the rationale for creating the dataset(s) clearly described?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Cell biology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.