Development and characterization of rabbit monoclonal antibodies that recognize human spermine oxidase and application to immunohistochemistry of human cancer tissues

Armand W. J. W. Tepper; Gerald Chu; Vincent N. A. Klaren; Jay H. Kalin; Patricia Molina-Ortiz; Antonietta Impagliazzo

doi:10.1371/journal.pone.0267046

Peer Review History

Original SubmissionDecember 3, 2021
7 Jan 2022 Decision Letter - Roberto Amendola, Editor PONE-D-21-38029Development and characterization of rabbit monoclonal antibodies that recognize human spermine oxidase and application to immunohistochemistry of human cancer tissuesPLOS ONE Dear Dr. Antonietta Impagliazzo, Thank you for submitting your manuscript to PLOS ONE. 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. ============================== The editor declares a major revision decision, based on both reviewer comments, and taking in strong consideration the points that myself raised in the first submission. In more details, reviewer #1 accomplished the benefit of your multiple monoclonal SMOX antibodies, but he invites the authors revising the list of references. The editor already invited the author to pose more attention on this point and ignoring it is certainly not a positive message for the editor and colleagues cited erroneously in the manuscript. Secondly, reviewer # 1 suggests to the authors providing evidences for a cross reaction with other model mammalian organisms beside human. It could not be an issue but it needs more experiments or comments. Reviewer #2 recognizes the work as solid with appropriate amount of experiments. Unfortunately, still the description of the statistical analysis is completing missing, as well the editor asked upon the original submission. Providing a paragraph in MM and mentioning the appropriate statistical analysis used in each experiment means which kind of statistical tool have been applied (student-T test, ANOVA, median range, etc). Moreover, all the Material and Methods section should be revised and described in more details (as an example, in immune-histochemistry, how have negative control been determined?). The editor still believe that your manuscript could be accepted, since both reviewers acknowledge the importance in the specific field of PA metabolism, but, once again, the editor invites primarily the corresponding author to work out on the points raised. ============================== Please submit your revised manuscript by Feb 21 2022 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. 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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. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. [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 Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: No ****** 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 Reviewer #2: 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 Reviewer #2: 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: The current manuscript by Tepper et al. describes the development of multiple monoclonal antibodies that recognize human spermine oxidase SMOX). As correctly pointed out in the manuscript, SMOX appears to be an important enzyme associated with multiple pathological conditions including many cancers. Current data suggest that SMOX plays a role in the etiology of multiple epithelial cancers and as suggest may represent a rational target for chemoprevention or early intervention. As such, the availability of highly specific, SMOX antibodies that can be used for multiple types of analyses would benefit the continued study of the role of SMOX in disease. The authors provide convincing data that they have developed sufficiently specific monoclonal antibodies to be used for Western blot, fluorescent microscopy, and IHC analyses of human SMOX. The data are presented in a clear and convincing manner and the tools described have been sufficiently tested for their selectivity in the various uses described. There are no major concerns regarding the presented data, or the conclusions made by the authors. However, there are a couple of minor points that should be addressed. 1) The authors state that, “In conclusion, the antibodies characterized here should meet the needs of most investigations involving hSMOX detection on gels, in solution, and in mammalian cells and tissues.” However, the only data presented are results with human SMOX. It would be helpful to demonstrate that the antibodies also recognize mouse or other model mammalian organisms SMOX. Although, this shouldn’t be an issue, since mouse and human SMOX share extensive homology, experience with polyclonal antibodies suggests that not anti-human SMOX antibodies are useful for detecting mouse or other mammalian SMOX. 2) There appears to be several errors in the Reference section. There is duplication of references, incorrect authorship, missing journal citations, etc. These must be corrected. Reviewer #2: Here it follows the evaluation of the MS (PONE-D-21-38029): “Development and characterization of rabbit monoclonal antibodies that recognize human spermine oxidase and application to immunohistochemistry of human cancer tissues” by Tepper et al. This paper deals about the production of a panel of high-affinity rabbit monoclonal antibodies against various SMOX epitopes and selected antibodies for different molecular analysis as immunoblotting, protein quantification assays, immunofluorescence microscopy and immunohistochemistry. Authors have identified the crucial epitops to generate specific antibody against SMOX with different features. The work is solid and the manuscript well written. A great amount of experiments and data are reported. It is an important contribution towards understanding of polyamine metabolism supplying new tools to following in vitro and in vivo SMOX expression. In particular, SMAB10 antibody allows following SMOX expression in different cancer tissues. The MS well adheres to the main points requested by Plos ONE journal policy but to major concerns need to be satisfied before acceptance. Major points: 1) Statistical analysis is completing missing, please provide a paragraph in MM and mention the appropriate statistical analysis used in each experiment and specify it in figure legends. 2) A comparison with other commercial antibodies would be beneficial for Plos ONE readership as: - Proteintech anti- SMOX Antibody 15052-1-AP \| - abcam anti-SMOX antibody ab213631 I recommend publishing of this MS on Plos ONE with the revisions outlined above. ****** 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 Reviewer #2: 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. https://doi.org/10.1371/journal.pone.0267046.r001
Revision 1
4 Mar 2022 Author Response Response to the reviewer of the manuscript: Development and characterization of rabbit monoclonal antibodies that recognize human spermine oxidase and application to immunohistochemistry of human cancer tissues Armand W.J.W. Tepper1; Gerald Chu2; Vincent N.A. Klaren1; Jay H. Kalin2; Patricia Molina-Ortiz3; Antonietta Impagliazzo1,4* n.b. The reference are to the lines in the revised manuscript with track changes. Reviewer #1 accomplished the benefit of your multiple monoclonal SMOX antibodies, but he invites the authors revising the list of references. The editor already invited the author to pose more attention on this point and ignoring it is certainly not a positive message for the editor and colleagues cited erroneously in the manuscript. Secondly, reviewer # 1 suggests to the authors providing evidences for a cross reaction with other model mammalian organisms beside human. It could not be an issue but it needs more experiments or comments. • We apologize for the number of errors in the previous list of references. We have checked and revised the reference section and hopefully provided the correct author's names and format. • Furthermore, we have reported the cross-reactivity of some of these antibodies with recombinant murine SMOX (Table 1 of the manuscript, line 389). We have added the figure of the WB in the supplementary information as well as an alignment of the human and murine sequence (line 382 and S6 Fig. line 384). We recognize that the quality of the blots is suboptimal, but unfortunately, we are unable to perform additional experiments due to the changes in circumstances. We hope, however, that this is convincing enough to illustrate the suitability of those mAbs for use in work with mouse models or tissues. Reviewer #2 recognizes the work as solid with appropriate amount of experiments. Unfortunately, still the description of the statistical analysis is completing missing, as well the editor asked upon the original submission. Providing a paragraph in MM and mentioning the appropriate statistical analysis used in each experiment means which kind of statistical tool have been applied (student-T test, ANOVA, median range, etc). Moreover, all the Material and Methods section should be revised and described in more details (as an example, in immune-histochemistry, how have negative control been determined?). • The statistical analysis is now clearly outlined in a paragraph added to the MM as the reviewer requested (row 253, Fig 5 legend at row 415). We have also added a S1 Table with the raw data and all the statistical parameters used in our analysis. • Furthermore, additional detail has been added to the MM section regarding the AlphaLisa (row 156) and Immunohistochemistry (row 205-219; 223-233) with clarification on the negative control determination. In brief, we selected Raji cells as negative control because publicly available transcriptomic data indicate a low basal hSMOX expression in this cell line (row 278), consistent with our own data. Raji cells were used to produce paraffin-embedded cell and xenograft samples to serve as a negative control in IHC (Fig 7). Attachments Attachment Submitted filename: Tepper et al_Response to the reviewer.docx https://doi.org/10.1371/journal.pone.0267046.r002
1 Apr 2022 Decision Letter - Roberto Amendola, Editor Development and characterization of rabbit monoclonal antibodies that recognize human spermine oxidase and application to immunohistochemistry of human cancer tissues PONE-D-21-38029R1 Dear Dr.Antonietta Impagliazzo, 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, Roberto Amendola, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. 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 Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. 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 Reviewer #2: Yes ****** 5. 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 Reviewer #2: Yes ****** 6. 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: The authors has sufficiently addressed my concerns and I believe if these monoclonal antibodies are made available to the polyamine community they will be of a major benefit. Reviewer #2: Here it follows the evaluation of the MS (PONE-D-21-38029): “Development and characterization of rabbit monoclonal antibodies that recognize human spermine oxidase and application to immunohistochemistry of human cancer tissues” by Tepper et al. In my opinion now, the manuscript is acceptable for publication. ****** 7. 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 Reviewer #2: No https://doi.org/10.1371/journal.pone.0267046.r003
Formally Accepted
14 Apr 2022 Acceptance Letter - Roberto Amendola, Editor PONE-D-21-38029R1 Development and characterization of rabbit monoclonal antibodies that recognize human spermine oxidase and application to immunohistochemistry of human cancer tissues Dear Dr. Impagliazzo: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Roberto Amendola Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0267046.r004

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