PONE-D-24-53200High-throughput 96-well plate-based porcine antibody isolation protocolPLOS ONE

Dear Dr. Crisci,

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the protocol been described in sufficient detail?

To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files.

The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses.

Reviewer #1: Partly

Reviewer #2: Yes

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3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.

Reviewer #1: No

Reviewer #2: Yes

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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: No

Reviewer #2: Yes

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5. Is the article 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 highlight any specific errors that need correcting in the box below.

Reviewer #1: No:  The author needs to be improved. Grammar and typos need to be corrected.

Reviewer #2: Yes

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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: In this work, Byrne et al., describe a protocol to isolate porcine specific IgG and IgA from serum, colostrum and milk in a high-throughput manner. The authors employ a 96-well plate setup in which they incubate samples with Protein G, CaptureSelect IgA, or Peptide M, followed by antibody isolation, concentration, and quantification. Overall, the manuscript is interesting, but several points need to be considered prior to publication.

The language of the manuscript needs to be carefully revised, since there are many grammatical errors and typos all over the work.

Ideally, the protocol included as supplementary material should have been integrated in the manuscript prior to submission to be able to appreciate how the whole manuscript looks.

Table 1 does not seem necessary, since this information is already mentioned in the text.

Figure 1. The influenza virus particle does not display the internal ribonucleoproteins.

Figure 2, ideally should depict a more detailed (but straightforward) scheme showing all major the steps of the protocol, including sample and resin plate prep, antibody isolation and concentration, and quantification.

It seems that the authors take a single point dilution to express antibody titers. This is usually inaccurate since results are highly dependent of the dilution used for calculating the optical density. An endpoint titer or area under the curve (AUC) value obtained from serial dilutions of the samples should be presented for the ELISA results.

It is unclear how serum HA-specific IgG can be so high (even higher) in the controls vs vaccinated animals. Likewise, how colostrum IgG and IgA are so high. Many studies using complete serum in different animal models show that responses in serum are usually specific, and show a clear distinction between controls and vaccinated animals.

The protein used in the methods is not described in the protocol not the ELISA method used for assessing the antibody levels. This information is important.

We recommend to remove filler words such as ‘clearly’, ‘dramatically’ (L151-153) throughout the manuscript.

Reviewer #2: In this article, Byrne et al. present a protocol that they established for high-throughput purification/isolation of immunoglobulins from porcine fluids that are prone to non-specific or high background binding to antigen, making these fluids unreliable for direct testing. The authors validated their method against influenza hemagglutinin.

This article merits publication because it is applicable towards other species (for which chromatography and Ig-specific resin or reagents are available); the authors validated their method for isolation of IgA after intranasal immunization, with this isotype predominant in mucosal immunity, protective against certain pathogens, and maternally transferred; they demonstrate efficacy of an intranasal vaccine targeting influenza hemagglutinin; they demonstrate that the species cross-reactivity of Ig-binding resin such as protein G extends to porcine antibodies.

I have some minor comments that may help the authors potentially improve the article. Apart from one overarching comment, the rest of the comments are organized into the sections as they appear in the text.

OVERALL

I feel that there are two topics that are equally important: the protocol itself for Ig isolation and immunization against influenza. However, it is unclear if the authors chose to prioritize one or the other or both. On the one hand, this is a “Lab Protocol” type article, with influenza HA presented as an antigen that helps validate the method (although demonstrating isolation of total Ig may have been sufficient). I see this reflected in the title and the abstract. On the other hand, the introduction leads with influenza, front and center, rather than opting for the structure of the abstract. Figure 3 also presents results on vaccine efficacy which give the impression of a “Research Article” type article. I think the article is publishable and acceptable as either.

The authors can choose to reinforce one aspect or the other as they see fit, to draw in a specific readership. I see advantages to both because a more general article would attract a broader readership because the method is applicable to other species and antigens. A more specialized article could have a larger impact within the authors’ direct field of study. However, the latter would require more details on the immunization, ELISA, and perhaps additional data analysis (see comments that follow).

INTRODUCTION

Line 63-64 – May be worthwhile to edit this sentence because we do not know what fraction of the antigen-specific antibodies are transferred passively to piglets as opposed to lost, and we also do not know if the antibodies are protective, only that they are antigen-specific. Neither transfer nor protection are directly studied here.

MATERIAL AND METHODS

I think details are missing, some important and some less important, about how the ELISA was performed. Since the introduction gives the impression that influenza is a major component of this article and this is backed up by the ELISA data, it would help to provide more details on the assay, if possible. It probably does not fit into the existing protocols.io page, but it would be helpful to document it in the main article.

I could be mistaken but the RESULTS section describe when the fluids were collected relative to parturition but not when they were collected relative to booster immunization. This is a factor for antibody titer and kinetics of development of an adaptive immune response.

The concentrations/dilutions of fluids used in the ELISA fit better here than solely in the Figure 3 legend.

Were the ELISAs all performed on the same plate/performed roughly at the same time/developed for the same duration (i.e., the time between substrate addition and stopping of the reaction)? This is related to the comment in the RESULTS section. If all specimens were tested on the same plate at the same time and developed for the same duration (roughly), then it makes it possible to judge the relative level of background binding from different fluids, the relative yield of antigen-specific Igs, compare them statistically, etc. I.e., multiple graphs in Figure 3 could be combined and additional statistical comparisons made. This information would be invaluable to steer the article toward more a "Research Article" type.

Protocols.io page – in the “Quantify Antibody concentration” section, was the “Sample Type” in the program set to “IgG” for example? It may help readers get a more accurate estimate of the protein concentration by instructing the program to use an extinction coefficient that is closer to that of polyclonal antibodies than the default extinction coefficient/sample type.

RESULTS

Whether from their own data or from the literature, could the authors comment on whether the protein G/peptide M/CaptureSelect resin have different affinities to different Ig subclasses (e.g., IgG1 vs. IgG2, etc.)? This is why it could be worthwhile to add more details about how the ELISA was performed (see comment in the MATERIALS AND METHODS section), in the event the secondary reagents were subclass-specific for example. Could help peers in the field.

Could the authors comment more and specify the differences in Ig yield instead of simply ranges in concentration? This could be an additional table. From the three fluids/compartments tested? Other comparisons: homeostatic levels of different Ig isotypes, any changes post-vaccination, compare the compartments, etc. This could help other researchers compare their yields with yours, gauge the binding capacity of the resins, etc.

Related to the comment above, for the data in Figure 3, since one of the objectives stated in the abstract and introduction was to remove background binding, is it possible to perform additional analyses such as comparing the OD between different fluids to gauge the level of background binding between fluids/compartments? An ANOVA would then be the appropriate test.

FIGURES

Figure 2 – This figure could benefit from more labels like in Figure 1. For example, the tube and pipette tip in the center is ambiguous. Otherwise, more details can be added in the legend.

MISCELLANEOUS

Please, correct some very minor grammatical and typographical errors below:

Line 30 – In “…to measure influenza-specific antibodies specific to pregnant and lactating pigs…”, the word “specific” appears twice in succession but with different meanings. Maybe replacing the “specific to” with “from immunized” could provide better context and clarity for the reader.

Line 57 – Please, delete the “an” before “influenza”.

Line 82 – Please, delete the “the” before “prior”. Replace “isolated” with “isolation”.

Figure 2 legend – two hyphens are missing for “HA specific” and “post vaccination”.

Figure 1 – Hyphen missing before “Derived” and another one missing before “specific”.

Step 35 of the protocols.io page – Correct the misspelled “yeild”.

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Reviewer #1: No

Reviewer #2: Yes:  Justin Tze Ho Chan

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High-throughput 96-well plate-based porcine antibody isolation protocol

PONE-D-24-53200R1

Dear Dr. Crisci,

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.

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Kind regards,

Victor C Huber

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #2: Yes

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2. Has the protocol been described in sufficient detail?

To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files.

The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses.

Reviewer #2: Yes

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3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.

Reviewer #2: Yes

**********

4. If the manuscript contains new data, have the authors made this data 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 #2: Yes

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5. Is the article 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 highlight any specific errors that need correcting in the box below.

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 #2: I am satisfied with how Byrne et al. responded to and addressed my suggestions and comments on the original draft of their manuscript. I have only minor comments and corrections to share with the authors for the revised manuscript. They are organized in the order that they appear in the text or in categories. However, even if this feedback is not 100% addressed, I would recommend/accept the manuscript for publication. Apologies for not catching or suggesting some of them earlier as I understand it may be too late for certain changes.

General

Since this is a “Lab Protocol” type article, involving a lot of methodology, it is natural to write in the passive voice, but perhaps alternating it with the active voice would make the manuscript more stimulating and increase readability in certain sections.

Introduction

Figure 1 legend – Since the figure may not be self-explanatory nor self-explanatory to everyone, readers may benefit from a more detailed legend, consistent with the Figure 2 legend. Every figure is the “the depiction” of something so perhaps the legend could be more informative and “standalone”, without readers having to pick up details from the main text.

Materials and Methods

I could be wrong, but bullet points are not a requirement for PLOS ONE for the methodology. Here, they are a bit odd and I am not sure if they are beneficial over paragraphs that are the format of organization everywhere else. However, I also have no way of seeing how bullet points would end up in the final typeset article so maybe it would not be so odd after all.

As a “Lab Protocol” type article, the materials are especially important and maybe the authors could consider integrating and organizing them into a categorized table, like in the Cell Press STAR methods “key resource table” format: https://www.cell.com/pb-assets/journals/research/cell/methods/table-template1-1699013648137.docx

The “List Materials” are especially random and could benefit from some organization and categorization.

Results

Line 164 – The “1:100 dilution” might contradict the “1:100 and 1:1000” on line 110.

Grammar

Line 64 – I am not familiar with what an “individual dose” refers to. Maybe a more fitting term would be “primary dose”, especially since the authors use it on line 163 as well.

Figure 2 legend – There are two hyphens missing: one on line 80 for “Pig-derived” and one on line 88 for “vaccine-induced”.

Line 95 – Replace “one” with “a”.

Line 163 – Add missing hyphens in “post-boost” and “post-prime”.

Figure 3 legend – On lines 179, 181 and 183, add the missing hyphens after “post”

Line 186 – Add the missing hyphen in “vaccine-induced”.

Protocols.io – Make title consistent with the one in the main text. Replace “insure” with “ensure” in Step 1, just like how the authors used “ensuring” in Step 5 and “ensure” in Step 23.

Thank you. I look forward to reading the final published work.

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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 #2: Yes:  Justin Tze Ho Chan

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