Progress in affinity ligand-functionalized bacterial magnetosome nanoparticles for bio-immunomagnetic separation of HBsAg protein

Leila Hatami Giklou Jajan; Seyed Nezamedin Hosseini; Mohsen Abolhassani; Masoud Ghorbani

doi:10.1371/journal.pone.0267206

Peer Review History

Original SubmissionDecember 20, 2021
2 Feb 2022 Decision Letter - Robert Chapman, Editor PONE-D-21-40019Dear editorial board of Plos One, Progress in Affinity Ligand-Functionalized Bacterial Magnetosome Nanoparticles for Bio-immunomagnetic Separation of HBsAg proteinPLOS ONE Dear Dr. Ghorbani, Thank you for submitting your manuscript to PLOS ONE. I am sorry for taking so long to return this manuscript to you. I have found it very difficult to recruit reviewers over the Christmas / New Year period and rather than delaying further waiting for reviewers, I have decided to proceed on the basis of the one external reviewer I have received and my own review of the manuscript (see below). 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. 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Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. 5. 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. 6. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files. Additional Editor Comments: As reviewer 1 points out, while the actual experimental results in this manuscript may not have been published elsewhere the work is very similar to previously published work. However, I think the work can be original in nature in its use of the platform for recovery of the antigen - to the best of my searching these results have not been published. I do agree with reviewer 1 that criteria 3-5 for publication are not yet met. 1. The nanoparticles are characterised by a single TEM image, a number DLS histogram, and an XRD showing Fe3O4. At a minimum, better TEMs, and the full DLS data (including intensity distributions) should be provided. 2. Attachment of the protein is shown by Bradford assay, but this does not prove covalent attachment. The control in these studies should not be ‘unmodified NPs’ but rather modified NPs without the DTT treatment (or similar). Likewise the FTIR data does not prove covalent attachment as the peaks that supposedly relate to the new bonds are present in the unmodified NPs too (and at best would only show the presence of the crosslinker, not evidence that this is how the protein is attached) It is hard to see how the following conclusion is supported by the data: “the extremely higher immobilization efficiency for BMs support can be related to superior features of biologically synthesized nanoparticles to synthetic magnetic nanoparticles and the use of optimum ligand density for immobilization process”. It could be just evidence that the free unbound protein has not been washed off the surface properly? 3. The immunoseparation studies are not well described or characterised. The recovery is shown by OD at 280nm but how do we know that all of this comes from the recovered antigen? ELISA at a single datapoint (concentrations not given) is shown in figure 3, but this should be done against a standard curve to show the concentration of active antigen that elutes from the column to support the OD calculation of recovery. A much more robust study of the immunoseparation is needed. 4. The language is not ‘clear, correct, and unambiguous’ and this should also be improved before publication. [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: Partly ******** 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: No ****** 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 authors have attempted to separate Hepatitits B surface antigen using magnetosomes. To me, it seems an extension of previously published article (https://doi.org/10.1016/j.bbrc.2007.03.156) where the authors developed magneto immuno PCR for the detection of Hbs ag. Further, the chemistry behind the linkage, bonding and separation are not detailed. Additionanlly, the authors should do more trials and comparative experiments to support their claim; rapid, sensitive, cost effectiveness and rapidness of this separation technology. Furthermore, the language must be improved. I would suggest the authors to improve the quality of the work and resubmit. ****** 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. https://doi.org/10.1371/journal.pone.0267206.r001
Revision 1
10 Mar 2022 Author Response Dear Dr. Robert Chapman, We appreciate you and the reviewers for your precious time in reviewing our paper and providing valuable comments. It was your valuable and insightful comments that led to possible improvements in the current version. The authors have carefully considered the comments and tried our best to address every one of them. We hope the manuscript after careful revisions meet your high standards. The authors welcome further constructive comments if any. Below we provide the point-by-point responses. All modifications in the manuscript have been highlighted in red. Sincerely, Masoud Ghorbani, D.V.M., Ph.D. Assistant Professor Pasteur Institute of Iran Journal 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. The manuscript was corrected according to PLOS ONE's style requirements. 2. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ. The ORCID ID of the corresponding author was placed in the Editorial Manager. 3. Please amend either the title on the online submission form (via Edit Submission) or the title in the manuscript so that they are identical. The title was amended in the on line submission. 4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. It was corrected as recommended as shown below The phrase “data not shown” was removed and replace with Fig 3, C and D). Fig 3. Identity and specificity characterization of purified anti-r-HBsAg Mab of P1C7. (A) The optical absorbance of purified protein. (B) ELISA distinguished specific binding to r-HBsAg protein, all used proteins including streptokinase (SK), interferon-gamma (IFN-2), and a mixture of BSA and Casein except for r-HBsAg exhibited inconsiderable absorbance. Anti-r-HBsAg Mab of P1C7 hybridoma cell line can detect r-HBsAg protein without cross-reactivity to others. (C) Coomassie Blue stained SDS PAGE (12%) of the purified Mab in reducing form: M (Ladder KD), lanes 1, 2, and 3 (Purified Monoclonal antibody (Mab) or (eluted protein from protein G column), lanes 4 and 5 (ascites fluid). (D) Western blot analysis of the r-HBsAg exposed to mAb of P1C7 hybridoma cell line, M (Ladder KD), lane 1, 2, and 3 (pure r-HBsAg) after developed with purified Mab. 5. 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. All images were adjusted and corrected as much as possible and resubmitted. The original uncropped and unadjusted images underlying the blot and the gel were reported in the submission’s figure 3. 6. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files. Tables were included in the body of the text as recommended Additional Editor Comments: As reviewer 1 points out, while the actual experimental results in this manuscript may not have been published elsewhere the work is very similar to previously published work. However, I think the work can be original in nature in its use of the platform for recovery of the antigen - to the best of my searching these results have not been published. I do agree with reviewer 1 that criteria 3-5 for publication are not yet met. We tried to correct all the issues quoted in criteria 3-5 in previous section. 1. The nanoparticles are characterised by a single TEM image, a number DLS histogram, and an XRD showing Fe3O4. At a minimum, better TEMs, and the full DLS data (including intensity distributions) should be provided. Concerning your comments, a better TEM was submitted to replace the previous one. Regarding the full DLS the below table was also added to the text to clarify how the XRD was calculated. Table 1: The obtained structural parameters of XRD analysis. The average crystallite size D of the particles was calculated from the Scherrer equation: D = Kλ/(βcosθ), where K is the Debye-Scherrer constant (0.89), λ is the X-ray wavelength (1.54 nm), β is the peak width of half-maximum (FWHM/2 = 2.5288 Å), and θ is the Bragg diffraction angle. Breifly, β×3.1416/180 →2.52887 × 3.1416 /180→ 0.0441; 2 Theta= 35.546 → θ= 17.773; Cos θ = 0.474363; D=Kλ/ (β cos θ)→ D= 0.94 × 1.54 / 0.0441 × 0.474363 → 69.199 nm. I (%) DXRD (nm) dhkl or (FWHM/2)(A⸰) 2θ (deg.) hkl 100 69.19 2.52887 35.546 311 2. Attachment of the protein is shown by Bradford assay, but this does not prove covalent attachment. The control in these studies should not be ‘unmodified NPs’ but rather modified NPs without the DTT treatment (or similar). Likewise the FTIR data does not prove covalent attachment as the peaks that supposedly relate to the new bonds are present in the unmodified NPs too (and at best would only show the presence of the crosslinker, not evidence that this is how the protein is attached) It is hard to see how the following conclusion is supported by the data: “the extremely higher immobilization efficiency for BMs support can be related to superior features of biologically synthesized nanoparticles to synthetic magnetic nanoparticles and the use of optimum ligand density for immobilization process”. It could be just evidence that the free unbound protein has not been washed off the surface properly? In respect to the Bradford assay and covalent attachment, I would like to emphasize that, we used cross-linker-activated nanoparticles as a standard to look for specific bonds that required us to use modified nanoparticles, and did not use unmodified nanoparticles due to non-specific bonds, because we were not interested in adsorption bonds, hydrogen bonds or van der Waals bonds. Regarding the links related to the FTR spectrum, it should be noted that in Figure 6 (C), the peaks are related to the 2362 wavelength of the monoclonal antibody. Whereas, the peaks 2854 and 2923 belong to the activated nanoparticles, both of which reappeared in the final product of conjugated antibody to nanoparticles. The wavelength of 1541 can be proof of the successful binding of nanoparticles to monoclonal antibodies from a specific site. Finally, in order to remove unwanted and non-specific bound proteins, the washing steps were performed very carefully and in several successive steps. Regarding the last part of this comment, it should be noted that the wash step was repeated at least three times to eliminate the possibility of unbinding proteins in the conjugated final product. Also, using stoichiometric calculations, by reading the adsorption of the fractions obtained from the wash and the total amount of antibody used at the beginning of the reaction, and also by reading the adsorption of the conjugated antibody, confirms the absence of unbinding proteins in the final product. 3. The immunoseparation studies are not well described or characterised. The recovery is shown by OD at 280nm but how do we know that all of this comes from the recovered antigen? ELISA at a single datapoint (concentrations not given) is shown in figure 3, but this should be done against a standard curve to show the concentration of active antigen that elutes from the column to support the OD calculation of recovery. A much more robust study of the immunoseparation is needed. With respect to your opinion, it should be noted that due to the use of specific antibodies as well as the identified antigen, due to the adsorption in OD 280 which is used to read the protein adsorption, the absorbed protein by antigen-specific antibodies, as well as complementary results in Figure 5 (where chromatographic and Western blot results are concerned), can be used to ensure that the protein obtained from the recovered yield is the antigen in question. At last, it should be noted that due to budget constraints, time, and laboratory facilities, it is not possible to perform further complementary experiments, and we hope that in the near future we will be able to elaborate more experiments to obtain more satisfactory results to report. 4. The language is not ‘clear, correct, and unambiguous’ and this should also be improved before publication. We had the language and the grammar checked by several interpreters and editors and did our best to remove the ambiguity and correct the grammar mistakes. Attachments Attachment Submitted filename: Response to Reviewers.doc https://doi.org/10.1371/journal.pone.0267206.r002
5 Apr 2022 Decision Letter - Robert Chapman, Editor Progress in Affinity Ligand-Functionalized Bacterial Magnetosome Nanoparticles for Bio-immunomagnetic Separation of HBsAg protein PONE-D-21-40019R1 Dear Dr. Ghorbani, 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, Robert Chapman, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): The authors have made no changes at all to this manuscript following the comments from the reviewers (one of which recommended a reject, and one of which asked for major revisions). They have also failed to address any of Reviewer 1's comments. 1) Characterisation of the nanoparticles: The authors claimed they replaced the TEM image but actually they just provided the same one inverted in the horizontal axis, with the brightness changed. Intensity distributions in the DLS were asked for and were not provided. The XRD does not help characterise the size and size distribution of the particles in solution. 2) Protein attachment: The FTIR does not prove covalent attachment, it just proves the presence of all of the components! The Bradford assay of the unmodified NPs are necessary if the authors want to claim (as they do) that the attachment of the protein is due to the SMCC linker and not due to non-specific interactions. This is not a very important point, and it would be fine if the authors tone down the conclusions drawn from this data - the antibody is clearly attached - the data just doesn't prove that its because of the SMCC linker. 3) Characterisation of the rHBsAb: The authors have pointed out that the SEC-HPLC analysis shows the recovered protein to be the rHBsAb. While data is not provided data to show that the protein is in its active form, this is perhaps not actually necessary to support the conclusions the paper draws. 4) No changes were made to address the language in the manuscript - the author's rebuttal on this point is just "we've had it checked and no changes are needed"! For these reasons I do not think the manuscript is 'of a high technical quality', and it remains very similar to previously published studies. There was an opportunity to greatly improve the paper on revision if the authors had been prepared to. However, it seems to me on reading their rebuttal that the actual conclusions they draw are supported by the data and for this reason it does meet PLOSone's publication criteria. https://doi.org/10.1371/journal.pone.0267206.r003
Formally Accepted
15 Jun 2022 Acceptance Letter - Robert Chapman, Editor PONE-D-21-40019R1 Progress in Affinity Ligand-Functionalized Bacterial Magnetosome Nanoparticles for Bio-immunomagnetic Separation of HBsAg protein Dear Dr. Ghorbani: 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. Robert Chapman Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0267206.r004

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