PONE-D-23-36865Generation and characterization of monoclonal antibodies against pathologically phosphorylated TDP-43PLOS ONE

Dear Dr. Zhang,

Thank you for submitting your manuscript to PLOS ONE, and personal apologies for the delay in getting back to you.Despite the requirement of two reviewers, I will go ahead with only one report since its constructive opinion summarizes all items that must be either amend or explain.After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jan 15 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Maria Gasset, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. "We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. 

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this brief manuscript, Zhang et al. describe the generation of novel antibodies directed against phospho-TDP-43 and provide compelling evidence of the antibodies´ validity detecting TDP-43 inclusions in situ. The data is clearly presented and the conclusions appear robust. The material presented here could be of interest and use in the field and the manuscript should be of interest to the reader. However, the manuscript could be further elaborated to provide a clear discussion positioning the novel antibodies with respect to the available ones and additional experiments could help to detail which species are really being detected. What follows is a list of issues which, in this reviewer´s opinion, could help improve the manuscript and categorize the described antibodies.

Major points:

#1. Please provide a clear description of the advantages of these novel antibodies with respect to the commercial ones, in particular in light of recent publications (Riemenschneider et al. https://doi.org/10.1186/s40478-023-01592-z).

#2. The major confusion raised while following the manuscript was trying to understand which species are specifically targeted by the antibodies. Although still open to debate, it could be concluded that phosphorylation of TDP-43 low complexity domain promotes insolubility (see da Silva et al. doi: 10.15252/embj.2021108443). In addition, CK1 phosphorylation may not be complete, which means that the antigens for immunization could correspond to aggregates containing mixtures of phosphorylated and non-phosphorylated TDP-43. In Figure 2, for instance, it is not clear if cell B supernatant is targeting aggregated (urea-soluble, in a broad way) rather than selectively phosphorylated TDP-43. In addition, Figure 4 suggests that the novel antibodies are targeting aggregated TDP-43. To clarify this, authors should demonstrate that aggregated, non-phosphorylated TDP-43 is not detected by the novel antibodies.

#3. Authors generate a double Ala mutant in positions 409/410 as a control of phosphorylation of these specific sites. Instead, authors should test the possible detection by the antibodies of a truncated version of TDP-43, where both 409 and 410 (and beyond) sites are not included.

Minor points:

#1. Please provide an explanatory scheme of the procedure followed to produce the antibodies and the specific epitopes which are targeted.

#2. Please include the sequence of the biotinylated peptides used in the dot-blots in Fig.2.

#3. Please include a loading control in the western blots (Calnexin, b-actin).

#4. Antibodies are used in 1:500 dilution in immunochemistry experiments. This appears too high. Please provide an explanation.

#5. Please show the uncropped gel in Fig. 1A, or at least the region covering molecular weights below 34 kDa. This is particularly relevant in Fig. 1B since the data with the doble Ala mutant appears crucial.

#6. In lines 219-220, reference to Fig 1B should instead be Fig 1C.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: Zhang et al review.pdf

Generation and characterization of monoclonal antibodies against pathologically phosphorylated TDP-43

PONE-D-23-36865R1

Dear Dr. Yong-Jie Zhang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Maria Gasset, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: