PONE-D-20-24214

A comparison of non-magnetic and magnetic beads for measuring IgG antibodies against P. vivax antigens in a multiplexed bead-based assay using Luminex® technology (Bio-Plex®200 or MAGPIX®)

PLOS ONE

Dear Dr. Longley,

Thank you for submitting your manuscript for review to PLoS ONE. After careful consideration, we feel that your manuscript will likely be suitable for publication if it is revised to address the points raised by the reviewers.  A significant number of topics need to be clarified and manuscript should be adjusted as suggested.   A Major concern was related to data analysis that should be revised as requested; perhaps controls using monoclonal antibodies could confirm the integrity of the exposed epitopes.  Finally, the authors should follow the policy of Plos One to share the raw data underlying their results.  Such policies help increase the reproducibility of the published literature, as well as make a larger body of data available for reuse and re-analysis. For your guidance, a copy of the reviewers' comments was included below. 

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We look forward to receiving your revised manuscript.

Kind regards,

Luzia Helena Carvalho, Ph.D.

Academic Editor

PLOS ONE

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2. Thank you for disclosing that RJL, TT and IM are inventors on patent PCT/US17/67926 on a system, method, apparatus and diagnostic test for Plasmodium vivax in your competing interest section. As this is an international patent application and not a granted patent, please revise this statement to say that RJL, TT and IM are inventors on "patent application" PCT/US17/67926.

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'This work was supported by the National Health and Medical Research Council Australia (https://www.nhmrc.gov.au/) (#1092789, #1134989 and #1043345 to IM and #1143187 to W-HT), the National Institute of Allergy and Infectious Diseases (https://www.niaid.nih.gov/grants-contracts/opportunities) (NIH grant 5R01 AI 104822 to JS) and the Global Health Innovative Technology Fund (https://www.ghitfund.org/) (T2015-142 to IM). Additional funding directly supporting field studies was from the TransEPI consortium (supported by the Bill and Melinda Gates Foundation, https://www.gatesfoundation.org/). RJL is currently supported by an NHMRC Early Career Investigator Fellowship (1173210). W.H.T. is a Howard Hughes Medical Institute-Wellcome Trust International Research Scholar (https://www.hhmi.org/programs/biomedical-research/international-programs, 208693/Z/17/Z).  We also acknowledge support from the National Research Council of Thailand. This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.'

We note that one or more of the authors are employed by a commercial company: CellFree Sciences Co., Ltd

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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

Reviewer #2: Yes

Reviewer #3: Partly

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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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

Reviewer #3: Yes

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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

Reviewer #3: Yes

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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 describe a systematic comparison of IgG antibody data as collected against a panel of 19 P. vivax antigens and human plasma samples from three endemic settings. This is an important study with the increase in use of the bead-based multiplex platform as performed by multiple groups worldwide, and the need to assess comparability among bead and platform variations. The study does well to directly compare variables of interest, but statistical presentation of results is only limited to correlation coefficients and p values inappropriately presented. The comparison should also include slope estimates for the regression models and p values corresponding to those.

Major concerns:

In the Introduction, the authors could do a better job of explaining the xMAP technology to the lay person unfamiliar with bead-based multiplex serology. Some suggestions are below, but review and imagine you’ve never heard of the multiplexing technology before when revising.

Table 1 and Table 2. For the benefit of the reader, for antigens that had been published on before, please include appropriate reference(s).

Throughout the paper, the authors state what appear to be p values for the r^2 correlation coefficient, which is inappropriate since r simply indicates goodness of fit for the regression model. For each comparison in the study, the authors should report both the r^2 as well as the slope, and can report on the p value of the slope. Additionally, if the slope deviates from 1.0, this is important information for the reader for generalizing if one bead type or platform would give consistently higher/lower MFI values. Instead of presenting all this information in the Figure panels, perhaps Tables (in a similar manner to Table 3) would make it easiest to digest. The authors should also state in the Methods what is criteria for ‘strong’ correlation.

In Conclusions, the authors accentuate the good correlation of responses between the various two comparators, but do not address potential reasons for when correlation (and slope) is not good. For example, why is MSP5 appear to have more issues than other Pv antigens? Many of the scatterplots in Figs 1 and 3 have very poor correlation and some points with differences >2 orders of magnitude, and this should be addressed.

Minor concerns:

- In title, spell out entire parasite name

- Throughout the document, if choosing to use the (R) symbol for company information, use consistently each time company is mentioned

- Abstract: instead of just mentioning responses were ‘strongly correlated’ also provide quantitative measures of correlation/difference (r^2 and slope estimates)

- Line 51-52: “such as a reduction in sample volume required and reduced laboratory time” if choosing multiple targets to assay for

- Line 57: “in more consistent findings among different studies”

- Line 59-62: if wanting to discuss the xMAP technology, need to be specific here about the IR dyes and pre-gating bead regions to allow multiplex data collection. Also need to mention the FlexMAP platform here. Provide a link to manufacturer’s website that explains this technology so readers can investigate for themselves.

- Line 62: explain what ‘coupling’ is

- Line 67: “Two different types of bead compositions are available…”

- Line 69-72: need to mention FlexMAP 3D system here

- Line 70-71: “offers advantages over the flow-based systems such as faster acquisition time” not true, and this is dependent on how many beads present in each assay well. Flow-based machines can actually read a plate faster than a MAGPIX

- Line 75-77: “A secondary aim was to demonstrate that this assay is highly reproducible in an independent laboratory through an external validation.” As currently worded, this isn’t an aim, but a pre-determined conclusion.

- Line 78-80: “however the large number of proteins assessed and consistent results obtained, suggest these findings should be generalizable for optimization of the multiplexed bead-based assay for other pathogens.” Even though there’s 19 antigens on your panel, they’re only to one pathogen. Not appropriate to extrapolate to the other myriad of human pathogens from this data alone.

- Line 80-82: “This is important in the context of the ongoing SARS-CoV-2 pandemic, as multiple laboratory assays based on Luminex technology are under development [6-8].” I get it that it’s really cool to talk about nCoV right now, but this sentence has nothing to do with your Pv malaria study and should be removed.

- Line 133-134: need to state here what would indicate that the beads were not stable

- Line 153-155, 162: Luminex recommends at least 35 beads acquired per region. Need to state here why 15 was chosen for this study.

- Line 266: “bead counts < 15”

- For Table 3 data, this is more of an “external comparison” rather than validation work

- Table 4: the majority of these factors (cost, time, etc.) are relative for an institution and the SOP being used, and this table should be removed since many of the statements are subjective

Reviewer #2: R. Mazhari and colleagues report the results from a straight-forward study that compared results using the original non-magnetic bead Luminex (100/200 – BioPlex) method with the newer magnetic bead-MAGPIX approach, by measuring antibody (Ab) levels to a series of P. vivax antigens. Although many researchers have conducted a few comparative assays when switching from the non-magnetic to magnetic format, to this reviewer’s knowledge, this is the first study to conduct an in-depth comparison between the two methods. Overall, the study is well described, the results clearly presented, and the results confirm what most investigators have assumed, namely, the two methods give very similar (but not identical) results. The study will provide reassurance to the research community that the two methods provide similar answers.

The following comments are meant to be helpful for improving the manuscript.

1. Methods: Line 153. “a 1:100 dilution of PE-conjugated Donkey …. was added.” More information is needed: What was the concentration used? How many ul were added? (e.g., the reagent (1 mg/ml) was diluted 1:1000 and 50uL was added to each well.”

2. Line 123-111124: “…, we optimized all protein concentrations …… .” This reviewer is not exactly sure what the statement means. Does it mean you coupled various concentrations of each antigen to beads, created a log-linear curve, and then selected a concentration of antigen that would give you a specific MFI after coupling using a 1:100 dilution of the plasma standard? This point is important for understanding Table 1, as the amounts listed do not appear to be “saturating” concentrations. It would also be useful the authors commented if it is possible to compare MFI between antigens, i.e., if 10,000 MF1 for antigen X means there are more Ab to X than 5,000 MFI to Ab Y? Some researchers report that if the beads are coupled with equal amounts of antigen, then comparison can be made across antigens (although I’m not sure I agree), but the question could be addressed.

3. It is surprising that the authors did not mention calibration or verification beads (standard), which I believe differ between BioPlex and MAGIX. Some mention of this would be useful. Were the same calibration beads (standards) used in the external validation study? If so, one might have expected even better agreement in SFig. 1 for MFI. Some mention of the influence (or lack thereof) of instrument calibration should be included.

4. In sFig. 1, it is somewhat difficult to interpret/compare the results, especially between antigens, because different Y- and X- axes were used. For example, for MSP1 the Y-Axis is from 100 to 10,000, but the X-axis is from 10 to 10,000. If similar results were obtained between the two assays, one should be able to draw a diagonal (45o) line, and half the data points would be above and half below. So, having the same X- and Y- axes is beneficial. It appears that higher MFI were obtained at CASE for antigens 112670, MSP7, and MSP8 (majority data points above the diagonal line), but lower values for 096995, PVx-001000, 094830. Do the authors have an explanation for this?

5. Converting raw data to arbitrary units using a standard curve is always difficult for readers to truly envision. It would be beneficial if a standard curve was included in the Supplemental Information section and some discussion provided on how MFI were converted to relative antibody units (RAU). That is, in sFig. 1 the MFI values range from 10 to >30,000 MFI (i.e., numbers normally found in the literature; readers will be able to identify with the numbers). In SFig. 2, data were transformed using the standard curve and range from 0.00001 to 0.1 arbitrary units. How did you get these numbers? Based on the patterns in paired figures, it appears 10,000 MFI equals ~0.01 RAU, right? This area of the manuscript would benefit from clarification. Based on the data provided and the reproducibility of the results across the various formats, it seems that reporting data as MFI would be beneficial, since Luminex data produced in all laboratories around the world are generated as MFI. Since the data using the standard curve is reported to be only slightly better, the authors might consider including a discussion/comment about the process of data transformation using R compared to simply using MFI.

6. SFig. 1 Y-axis is labeled Case; whereas, in sFig. 2 it is labeled CWRU. The labels should have the same nomenclature.

Minor comments:

1. Abstract: Technically, the word data is a plural term (datum singular). So, Line 34 should read “Data are lacking …..”. However, the world may be changing in how this term is perceived.

2. Line 49: “…. Has been a rapid uptake of Luminex ……” The word “uptake” is colloquial, so suggest changing to another term, such as, increased, development, advancement, etc.

3. Line 54: Do Plasmodial parasites really express hundreds of thousands of proteins?

Just comments for the authors (FYI: no response needed)

1. Line 162: Our biostatisticians also calculated that 15 beads was the lowest number of beads needed to provide representative data. Alas, most investigators think that bead counts need to be ≥100. So, your manuscript will help set the record straight.

2. We have also found that antigen-coupled beads are quite stable. We have used the same batch of coupled beads, without loss of MFI, for over 10 years. We have also found that antigens tend to be more stable after they are coupled than if they are re-frozen and recoupled.

Reviewer #3: The authors presented a technical- descriptive paper which aimed to compared two different tools (beads) applied to serological multiplex methods. This bring interesting results that could help cutting steps on laboratory work and to choose the better technique for serological studies. The authors cited previews studies where beads-based multiplex assays have been used, suggesting that kind a technical is going to be the future for serological studies, as screening to evaluation of the antibody responses. But the standard immuno-assay (ELISA) is still a standard protocol to this kind of evaluation. Also, the use of a coupling beads needs more validations regarding the integrity of the protein conformation, once the coupled protein could hide some important epitope to antigen recognition by antibodies. Taken this and to make the present study afford to publication, I’d like to make some suggestions:

1. Show the standard curve for both type beads in the main manuscript. This is a big step to select the optimized antigen concentrations of the protein and beads necessary the assay. Also, will be interesting show the detail how MFI is converted to relative antibody units (RAU) using protein-specific standard curve data.

2. As the author are presenting a technical paper, showing a technique that could replace the ELISA, I suggest show the antibody titles do not change for coupled and non-coupled antigen for both type beads, using for example, the standard ELISA for at least an antigen with weak correlation (PVX_003770), and with an antigen with a high correlation, and decenter controls. Those technique aim to show the antibodies titles, which cannot be significant different compare with a standard immunological assay (ELISA).

3. I’m concern about the protein conformation preservation when it is coupled. Maybe a assay using monoclonal antibodies for which recognize the specific epitopes should be interesting to show that protein conformation are being preserved.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: 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.]

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PONE-D-20-24214R1

A comparison of non-magnetic and magnetic beads for measuring IgG antibodies against Plasmodium vivax antigens in a multiplexed bead-based assay using Luminex® technology (Bio-Plex®200 or MAGPIX®)

PLOS ONE

Dear Dr. Longley,

Thank you for submitting your manuscript for review to PLoS ONE. After careful consideration, we feel that your manuscript will likely be suitable for publication if it is revised to address few points raised now by the reviewer. We therefore invite you to revise your manuscript paying close attention to the specific points detailed by the reviewers. 


Please submit your revised manuscript by November 30. 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Luzia Helena Carvalho, Ph.D.

Academic Editor

PLOS ONE

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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: (No Response)

Reviewer #3: All comments have been addressed

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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

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: 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

Reviewer #3: 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: (No Response)

Reviewer #2: The authors have made changes in the manuscript that enhance its clarity and addressed the issues raised by the reviewers. The study clearly makes the point that a correlation exists among the data generated using difference beads-based systems. The authors might consider the following suggestions:

1. The authors added the sentence: “This is expected given the conversion, based on the standard curve generated with a plasma pool from immune PNG donors…..” Was the standard curve run on each plate? If so, please add this information to the text.

2. Supporting Fig. 1 shows the linearity of the dilution curve. However, it is not totally clear how one obtains relative arbitrary unit (RAU) from the curve (MFI vs S1, S2, etc.). Could the authors please add information to the figure legend on how to convert the data from MFI to RAU?

3. Supportive Fig. 2 – YEAH! This figure shows the raw MFI and that subsequent statistical refinements not really needed. All readers will appreciate this figure.

4. Supportive Fig. 3 – shows the relationship of the adjusted values and documents the association after statistical adjustments. Interestingly, adjustments changed the correlations insignificantly. Nice to see the comparison.

5. Supplemental Figures 3 and 4 appear to be the same – comparison of RAU at CWRU and WHEI. Maybe the wrong figure was uploaded into the copy I received. Supportive Figure 4 doesn’t show changes in MFI over time. The authors need to check sFig. 4.

Reviewer #3: (No Response)

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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: Yes: Eric Rogier

Reviewer #2: Yes: Diane Wallace Taylor

Reviewer #3: 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.

A comparison of non-magnetic and magnetic beads for measuring IgG antibodies against Plasmodium vivax antigens in a multiplexed bead-based assay using Luminex® technology (Bio-Plex®200 or MAGPIX®)

PONE-D-20-24214R2

Dear Dr. Longley,

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,

Luzia Helena Carvalho, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: