A high-throughput microsphere-based immunoassay of anti-SARS-CoV-2 IgM testing for COVID-19 diagnostics

Dayu Zhang; Tianyang Xu; Eric Chu; Aiguo Zhang; Jinwei Du; Michael Y. Sha

doi:10.1371/journal.pone.0248444

Peer Review History

Original SubmissionMarch 22, 2021
10 May 2021 Decision Letter - Chandrabose Selvaraj, Editor PONE-D-21-09355 A High-throughput Microsphere-based Immunoassay of Anti-SARS-CoV-2 IgM Testing for COVID-19 Diagnostics PLOS ONE Dear Dr. Sha, 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. Please submit your revised manuscript by Jun 21 2021 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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Please review the manuscript in accordance with the reviewer's comments and submit, along with reference checks. [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 Reviewer #2: Yes Reviewer #3: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: 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: No Reviewer #2: Yes Reviewer #3: No ****** 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 ******** 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: This article is well written. There are some missing informations and discussion. Here are my suggestions: 2.3 Assay procedure What are the two blank wells used to calculate the ratio ? You should describe if it is a human sample, or a synthetic sample. Cut-off value is set to 45. You should indicate how this cut-off has been determined (same comment in the clinical performance section). 3.1 Clinical Performance Table 1: please replace “IgG Positive” / “IgG Negative” by “IgM Positive” / ”IgM Negative” Healthy donors data should be presented and discussed. Data ROC curve and AUC analysis should be performed. The methodology used to determine the Cut-off value should be presented. Instead of “data not shown”, data generated with matched serum and EDTA plasma should presented to demonstrate that EDTA plasma can be used. 3.3 Precision “Inter-assay reproducibility (between-run precision) was evaluated by testing negative sample and positive sample on five separate runs “ Please correct: “Inter-assay reproducibility (between-run precision) was evaluated by testing one negative sample and two positive samples on five separate runs “ You should indicate in table 4 that ratio are presented. It would be interesting to give additional characterization of serum#36 and Serum#32 (for instance the level positivity on a reference test). I would suggest to mention the stability of the developed reagents. 4. Discussion You should address a major question: Why do you use Luminex technology in a simplex mode ? You might want to present some preliminary data of an IgM and IgG kit. Reviewer #2: In this study, a high-throughput immunoassay of anti-SARS-CoV-2 IgM antibody was carried out on Luminex Magpix platform. A total of 42 serum samples from patients with positive COVID-19 were collected. This study has certain value for the control of SARS coronavirus. However, the authors should declare the creativity of this work and compare it in depth with other standard IgM tests。 Reviewer #3: The anti-SARS CoV2 IgM assay described is rather straight forward one. The investigators have used commercially available recombinant SARS CoV2 Spike protein 1 (RBD) and conjugated with commercially available microsphere and all reagents used in the assay were commercial ones. Some of the points that need clarification are: 1. What was the source of Covid positive and covid negative patient sample? How as Covid positivity or negativity determined? This information is crucial since all conclusions on performance are based on these samples. Were the patients RT-PCR positive or seropositive? If latter, what was the methodology used and the status of seropositivity (igM or IgG or both?). 2. As per section 3.4, there was data on igG positivity and IgM negativity for 10 samples from another commercial kit. What about others (totally 42 SARS CoV2 positive used). 3. The study was designed to test for SARS CoV2 IgM, but Table 1 shows data for IgG. What about IgM? [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.0248444.r001
Revision 1
25 Jun 2021 Author Response 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: This article is well written. There are some missing informations and discussion. Here are my suggestions: 2.3 Assay procedure What are the two blank wells used to calculate the ratio? You should describe if it is a human sample, or a synthetic sample. The blank wells consist of 3 µL of S1 RBD protein coated magnetic beads, 87 µL of BlockAid™ Blocking Solution, and 10 µL PBS-1% BSA. This information was added to the manuscript’s Methods 2.3 Assay procedure. . Cut-off value is set to 45. You should indicate how this cut-off has been determined (same comment in the clinical performance section). The cut-off value was calculated based the ratio distribution of 162 COVID-19 negative samples tested. This information was added to the manuscript ‘s Methods 2.3 Assay procedure. . 3.1 Clinical Performance Table 1: please replace “IgG Positive” / “IgG Negative” by “IgM Positive” / ”IgM Negative” Have replaced. Thank you. Healthy donors data should be presented and discussed. Data ROC curve and AUC analysis should be performed. The methodology used to determine the Cut-off value should be presented. According to your suggestion, the ROC curve is drafted and shown in Figure 2. The Area under the ROC Curves (AUCs) are 0.85, 0.67, and 0.38 for samples collected on 0-7 days, 8-14 days, and 2-8 weeks from symptom onset, respectively. This information was added to the manuscript. Instead of “data not shown”, data generated with matched serum and EDTA plasma should presented to demonstrate that EDTA plasma can be used. According to your suggestion, the data is now presented in Table 2. 3.3 Precision “Inter-assay reproducibility (between-run precision) was evaluated by testing negative sample and positive sample on five separate runs “ Please correct: “Inter-assay reproducibility (between-run precision) was evaluated by testing one negative sample and two positive samples on five separate runs “ Have corrected. Thank you. You should indicate in table 4 that ratio are presented. It would be interesting to give additional characterization of serum#36 and Serum#32 (for instance the level positivity on a reference test). Table 4 (now Table 5) now indicated that ratios are presented. I would suggest to mention the stability of the developed reagents. we have tested the reagent stability and confirmed the shelf life of the kit is about 12 months. 4. Discussion You should address a major question: Why do you use Luminex technology in a simplex mode ? You might want to present some preliminary data of an IgM and IgG kit. Great question. We do have another kit for anti-SARS-CoV-2 IgG detection. Luminex Magpix is used in a simplex mode due to specific customer needs. As a positive IgG test indicates protection against infection (i.e., sought after by people who are vaccinated) and a positive IgM test indicates recent infection (i.e., required by travelers flying to China), these two tests were rarely purchased together by our customers. Reviewer #2: In this study, a high-throughput immunoassay of anti-SARS-CoV-2 IgM antibody was carried out on Luminex Magpix platform. A total of 42 serum samples from patients with positive COVID-19 were collected. This study has certain value for the control of SARS coronavirus. However, the authors should declare the creativity of this work and compare it in depth with other standard IgM tests。 This is a microsphere-based immunoassay. It is semi-quantitative assay and can give a quantitative IgM (MFI) for each patient. This is particle important when we discuss this with vaccinated individual. We have demonstrated infected individual has same amount IgM for N protein and S1 protein. However, for recovered and vaccinated individual, anti SARS-CoV-2 N IgM and S1 IgM has different amount which it can be distinguished each other (our different manuscript). It can run 96 samples each time during 3 hrs. It is quite different to LFI in the throughput and accuracy. Reviewer #3: The anti-SARS CoV2 IgM assay described is rather straight forward one. The investigators have used commercially available recombinant SARS CoV2 Spike protein 1 (RBD) and conjugated with commercially available microsphere and all reagents used in the assay were commercial ones. Some of the points that need clarification are: 1. What was the source of Covid positive and covid negative patient sample? How as Covid positivity or negativity determined? This information is crucial since all conclusions on performance are based on these samples. Were the patients RT-PCR positive or seropositive? If latter, what was the methodology used and the status of seropositivity (igM or IgG or both?). These samples were tested by FDA EUA IgM assay DIAZYME SARS-CoV-2 IgG/IgM CLIA kit and the patient were also confirmed by FDA EUA qPCR. We noted in section 2.2 Reagents and patient samples that COVID-19 patient serum samples were acquired from ProMedDx. We noted in section 3.1 Clinical Performance that the patients are RT-PCR positive. 2. As per section 3.4, there was data on igG positivity and IgM negativity for 10 samples from another commercial kit. What about others (totally 42 SARS CoV2 positive used). The patient samples were acquired based on RT-PCR result thus not all samples were tested for antibodies. 3. The study was designed to test for SARS CoV2 IgM, but Table 1 shows data for IgG. What about IgM? Sorry, it was an error. The table was corrected. The results shown are IgM tests instead of IgG. Sorry for the confusion. Attachments Attachment Submitted filename: reviewers questions and answers.docx https://doi.org/10.1371/journal.pone.0248444.r002
19 Jul 2021 Decision Letter - Chandrabose Selvaraj, Editor A High-throughput Microsphere-based Immunoassay of Anti-SARS-CoV-2 IgM Testing for COVID-19 Diagnostics PONE-D-21-09355R1 Dear Dr. Sha, 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, Chandrabose Selvaraj, 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 Reviewer #3: 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: (No Response) Reviewer #2: Yes Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) Reviewer #2: Yes Reviewer #3: N/A ****** 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: (No Response) 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: (No Response) 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: (No Response) Reviewer #3: Revision has answered all the reviewer questions and provided clarifications where needed. There are no outstanding issues to be looked onto. https://doi.org/10.1371/journal.pone.0248444.r003
Formally Accepted
24 Aug 2021 Acceptance Letter - Chandrabose Selvaraj, Editor PONE-D-21-09355R1 A High-throughput Microsphere-based Immunoassay of Anti-SARS-CoV-2 IgM Testing for COVID-19 Diagnostics Dear Dr. Sha: 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. Chandrabose Selvaraj Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0248444.r004

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