Rapid Quantification of SARS-CoV-2 Neutralising Antibodies Using Time-Resolved Fluorescence Immunoassay
Round 1
Reviewer 1 Report
This study shows an evaluation of a surrogate virus neutralization test (sVNT) kit that quantifies neutralizing antibodies for SARS-Co-V2. The authors used WHO standard immunoglobulin preparations to show the linearity, sensitivity, and specificity of the kit. The results show excellent performances in a short test time 915 minutes). This kit is expected to provide huge potential for measuring immunity status on large scale.
There are some comments and questions as follows.
(1) at line 82, the unit of dilution concentration is not clear from a typo.
(2) More specific information on the instruments such as make and nationality would be helpful. For instance, about the LS-1100 and LS-21100 at lines 92 and 93, and GraphPad Prism software at line 99.
(3) Regarding the ELISA evaluation, its purpose and role are not clear in this study. Even though LOD tendency is mentioned in lines 135 – 141, the results seem natural and do not have any specific meaning to this study. A further clear explanation would be helpful.
(4) Regarding the PL-NAb tests, how many samples for each dilution were repeated?
(5) This study provides the correlations between the tested NAb, and reported NAb and anti-RBD. What were the correlations with other reported antibodies? Is there any reason that the other antibodies were not shown?
Sincerely,
The reviewer.
Author Response
We thank the reviewer for the kind comments and for the careful analysis of our first submission. We have addressed the questions of the reviewer as described below. We hope that these additions and clarifications satisfy the concerns raised.
(1) at line 82, the unit of dilution concentration is not clear from a typo.
This was introduced after text format change to the template and missed. It has been corrected to the correct units and is now on line 100 after modifications and additions to the introduction section changed the line numbering.
(2) More specific information on the instruments such as make and nationality would be helpful. For instance, about the LS-1100 and LS-21100 at lines 92 and 93, and GraphPad Prism software at line 99.
This oversight of ours has been corrected to add Lansion Biotechnology (lines 111-112) and details of GraphPad Prism software added to lines 118-119. Line numbering has changed due to earlier text additions to the introduction.
(3) Regarding the ELISA evaluation, its purpose and role are not clear in this study. Even though LOD tendency is mentioned in lines 135 – 141, the results seem natural and do not have any specific meaning to this study. A further clear explanation would be helpful.
This additional information on ELISA was an oversight of ours and we thank the reviewer for identifying this omission. We added a sentence to the methods clarifying the use of ELISA (lines 97-98) and a statement within the results that assist with comparison of ELISA to PL-Nab (lines 162-164). We have also added a sentence to the discussion that reflects further on this analysis (lines 255-256).
(4) Regarding the PL-NAb tests, how many samples for each dilution were repeated?
This information was already in the legend of figures 2 (now lines 209-210) and 3 (now lines 215-216) – triplicate determinations were done. However, we have now added this to the figure 3 legend (line 218) and the methods section for clarity (line 109).
(5) This study provides the correlations between the tested NAb, and reported NAb and anti-RBD. What were the correlations with other reported antibodies? Is there any reason that the other antibodies were not shown?
We added a sentence in discussion (line 262-266) to explain why this analysis was not done. The prior part of the discussion (now lines 257-263) had already explained the scientific reasons why the analysis is not likely to add to what we have determined. Effectively, correlations to NAb and anti-RBD are reflecting what is actually measured by the PL-NAb test and extrapolating that to other parameters such as anti-S and anti-N are not likely to add much as similar correlations are expected. In fact, reported values for these additional parameters are broadly similar to anti-RBD. Therefore we think that the correlations shown are sufficient to understand and interpret these data obtained with PL-NAb and match well with reported information and immune correlates of protection for SARS-CoV-2.
Reviewer 2 Report
The authors have presented a novel method for quantification of SARS-CoV-2 neutralising antibodies which is based on time resolved fluorescence immunoassay. I think the results and the methodologies discussed in this paper are very interesting and have translational medicine applications. I have the following concerns about the paper and I hope the authors can improve the paper after addressing them:
1. The introduction needs to be improved and review the current status and importance of the detection assays for SARS-CoV-2 NAb.
2. Figure 1 can be improved graphically.
Author Response
The authors have presented a novel method for quantification of SARS-CoV-2 neutralising antibodies which is based on time resolved fluorescence immunoassay. I think the results and the methodologies discussed in this paper are very interesting and have translational medicine applications. I have the following concerns about the paper and I hope the authors can improve the paper after addressing them:
We thank the reviewer for the positive comments made and careful analysis of our paper. We have made corrections and alterations according to the comments made as follows.
The introduction needs to be improved and review the current status and importance of the detection assays for SARS-CoV-2 NAb.
We have significantly modified the paragraph 2 of the introduction to improve this section and to carefully reflect the reviewer comments. These are shown in lines 32-60 within the introduction. These changes also required the addition of two new supporting references 10 and 11, therefore we also modified the reference list to accommodate these. As a result of these changes to the introduction, we feel that this section has improved to reflect the importance and current status of NAb testing for SARS-CoV-2.
Figure 1 can be improved graphically.
Thank you for suggesting this. We have made changes to the figure and modified the graphics to improve ACE-2 and RBD schematics shown in the new Figure 1 on page 5. Some minor text additions to explain the figure more carefully have also been added. We hope that the new version satisfies the reviewer concerns about the original figure graphics.
Round 2
Reviewer 1 Report
All the comments and questions from this reviewer have been cleared in the revised manuscript. This manuscript is recommended to be published.
