SARS-CoV-2 Rapid Antigen Test Based on a New Anti-Nucleocapsid Protein Monoclonal Antibody: Development and Real-Time Validation

Round 1

Reviewer 1 Report

Comments to the Author

In this manuscript, Fabiana Fioravante Coelho et al. describe the development and validation of an Ag-RDT, during an outbreak of the Omicron variant, including the characterization of a new monoclonal antibody (anti-DTC-N 1B3 mAb) that recognizes the Nucleocapsid protein (N). While the manuscript is well-written and presents an interesting conclusion, there are several major and minor comments.

Major：

Point 1. Despite the continuous mutations in SARS-CoV-2, the occurrence of mutation sites in the N protein is relatively limited, and most commercially available antigen detection test kits show little impact on the sensitivity of SARS-CoV-2 variants. To better understand the rationale behind selecting truncated N protein, it would be helpful if you could clarify the advantages and specific reasons for choosing this approach.

Point 2. In your research, the sensitivity of the antigen detection test kit for SARS-CoV-2 was reported as 95.2% at Ct ≤ 25, whereas most commercially available kits have a sensitivity of approximately 100% under the same conditions. Identifying the factors contributing to this variance will help readers interpret the significance of your study accurately.

Point 3. It is crucial to include a comparison with more antigen detection test kits produced by various biotech companies from different regions, such as the USA, China, Europe, etc. Expanding the analysis to include a more diverse range of commercially available kits will provide a more comprehensive evaluation and enhance the overall relevance of your study.

Minor：

Point 1. Table 2 and Figure 2B contain redundant information. To avoid unnecessary repetition and make the presentation more concise, I recommend retaining one of these elements while ensuring that all essential data and findings are adequately represented.

 

Minor editing of English language required.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

2. Materials and Methods - Weaknesses and Limitations:

While our study aimed to contribute valuable insights into the detection and characterization of SARS-CoV-2 and its variants, several limitations and potential sources of bias should be acknowledged:

1.            Sample Size and Diversity: Our study was based on a specific population from a single region, potentially limiting the generalizability of our findings. A larger and more diverse sample could enhance the external validity and applicability of our results.

2.            Assay Development and Validation: Developing accurate and reliable diagnostic tests is challenging. While we meticulously validated our diagnostic assay, further independent validation studies could enhance the robustness of our findings and confirm the diagnostic accuracy in diverse settings.

3.            Variable Sensitivity: Our diagnostic test's sensitivity is a critical parameter for its clinical utility. While our assay exhibited high sensitivity, it's important to acknowledge that false negatives might occur, especially in cases of low viral loads. Optimizing sensitivity for varying viral loads could enhance its effectiveness.

4.            External Validation: External validation of our diagnostic test using independent samples is essential to verify the reproducibility of our findings in different populations and settings, reducing the risk of overestimation.

5.            Possible Bias: The potential for bias in sample collection, even with rigorous protocols, could impact the representativeness of our study population. Strategies to minimize selection bias during sample collection would bolster the validity of our results.

6.            Follow-Up Duration: The follow-up period of 17 days for monitoring COVID-19 patients may not capture the entire spectrum of disease progression and immune responses. Longer follow-up durations could provide a more comprehensive understanding of disease dynamics.

7.            Cross-Reactivity and Molecular Variability: While our diagnostic test showed specificity for SARS-CoV-2, potential cross-reactivity with other related pathogens was not exhaustively investigated. Moreover, the potential for detecting emerging variants, such as those associated with VOCs, warrants continuous surveillance and adaptation of the diagnostic test.

8.            Long-Term Stability: We acknowledge that the long-term stability of diagnostic tests is crucial for their clinical use. Further studies evaluating the stability of our assay components over extended periods would provide confidence in its reliability.

Needs polishing and tuning the tone of the paper.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

I read with great interest the paper authored by Coelho et al. entitled "SARS-CoV-2 rapid antigen test based on a new anti-nucleocapsid protein  monoclonal antibody: development and real-time validation".

The manuscript is metodologically correct and with good scientific soundness.

I really think that a new antigen rapid diagnostic test may constitute alternative tools for COVID-19 point-of-care diagnosis and epidemiological surveillance.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Accept in present form.