Development and Validation of Competitive ELISA for Detection of H5 Hemagglutinin Antibodies

Round 1

Reviewer 1 Report

Manuscript poultry-2410559 entitled “Development and validation of a recombinant-H5 hemagglutinin-based competitive ELISA for serodiagnosis of influenza A virus H5 subtype antibodies”. Please notice the following:

General view: The manuscript highlighted a great laboratory hypothesis with the actual detailed steps for development and validation. The manuscript was expressed properly. 

Title: Clear to a greater extent but preferred to be more concise as follows: “A Recombinant-H5 Hemagglutinin-based Competitive ELISA Development and Validation against Influenza A H5”.  

Abstract: Clear and informative. A certain degree of copyediting and rephrasing is advisable to enhance readability and understanding.

Introduction: Improperly arranged into four paragraphs. The introduction section must be re-arranged into only three paragraphs i.e., 1. Introduction 2. Significance of the study, and 3. Aim of the study. Some modifications have to be carried out to enhance the readability and understanding of the text.

The aim: Clear and informative.

Materials and Methods: Clear to a greater extent. Please provide the statistical model used for the analysis. Some modifications have to be carried out to enhance the readability and understanding of the text.

Results: Novel, clear, and informative. A certain degree of copyediting and rephrasing is advisable for simplification and to enhance readability and understanding.

Discussion: Clear, informative, and contribute to knowledge with a good level of speculation and a high level of comparison. Some modifications have to be carried out to enhance the readability and understanding of the text.

Conclusion: Perfred to be a separate section instead of a paragraph by the end of the discussion. Clear but a few modifications have to be carried out to enhance the readability and understanding of the text.

Authors’ contributions: Clear and informative.

Acknowledgment: Clear and informative.

Funding: Clear and informative.

References: Sufficient as 32.3% (11 out of 34) were published in the past five years.

Tables: Well organized and presented.

Figures: Well organized and presented.

The authors expressed their novel work using good language and grammar. The manuscript was expressed properly. A certain degree of copyediting and rephrasing is advisable for simplification and to enhance readability and understanding throughout the manuscript.

Author Response

Reviewer #1

Title: Clear to a greater extent but preferred to be more concise as follows: “A Recombinant-H5 Hemagglutinin-based Competitive ELISA Development and Validation against Influenza A H5”.  

The title of the manuscript was modified and now reads as follows: Development and validation of competitive ELISA for detection of H5 hemagglutinin antibodies.

Abstract: Clear and informative. A certain degree of copyediting and rephrasing is advisable to enhance readability and understanding.

We modified parts of the abstract as suggested so that it is clearer (highlighted in yellow)

Introduction: Improperly arranged into four paragraphs. The introduction section must be re-arranged into only three paragraphs i.e., 1. Introduction 2. Significance of the study, and 3. Aim of the study. Some modifications have to be carried out to enhance the readability and understanding of the text.

Paragraphs were rearranged into 3 as suggested. The following sentence was added at the end of third paragraph to describe the aim of the study.

In this study, a cELISA immunoassay based on mAb #10 (F37H5N1-56) and recombinant H5 HA antigen derived from a Eurasian strain A/Teal/Germany/Wv632/2005 (H5N1) (clade EA-nonGs/GD) was developed, validated, and evaluated with high specificity, sensitivity, and low variability. In particular, the developed cELISA is capable in detecting antibodies in serum samples collected from different species animals that were infected with currently circulating clade 2.3.4.4b H5Nx viruses.

Materials and Methods: Clear to a greater extent. Please provide the statistical model used for the analysis. Some modifications have to be carried out to enhance the readability and understanding of the text. The following paragraph on statistical analysis was added.

2.9. Statistical analysis

The rec-H5 antigen-based cELISA was validated using ROC analysis and calculated diagnostic performance, which included the determination of sensitivity, specificity, and threshold cut-off using SigmaPlot version 14.0 (Systat Software, San Jose, CA) for statistical analysis. The measurement of agreement between the gold standard HI test and the cELISA tests was assessed using Cohen’s kappa (κ) test. Interpretation of the given κ value utilized the criteria laid out by Landis and Koch [27]. κ ≤ 0.00 was designated as poor agreement, 0.00 < κ ≤ 0.20 slight agreement, 0.21 < κ ≤ 0.40 fair agreement, 0.41 < κ ≤ 0.60 moderate agreement, 0.61 < κ ≤ 0.80 substantial agreement, and 0.81 < κ < 1.00 almost perfect agreement.

Results: Novel, clear, and informative. A certain degree of copyediting and rephrasing is advisable for simplification and to enhance readability and understanding.

The results section was edited for clarity. The edited part is highlighted in yellow.

Discussion: Clear, informative, and contribute to knowledge with a good level of speculation and a high level of comparison. Some modifications have to be carried out to enhance the readability and understanding of the text.

The discussion section was edited for clarity. The edited part is highlighted in yellow.

Conclusion: Preferred to be a separate section instead of a paragraph by the end of the discussion. Clear but a few modifications have to be carried out to enhance the readability and understanding of the text.

The conclusion section was modified and split into 2 sections. The edited part is highlighted in yellow.

Reviewer 2 Report

Dear colleagues

The following review article entitled “Development and validation of a recombinant-H5 hemagglutinin-based competitive ELISA for serodiagnosis of influenza A virus H5 subtype antibodies” is well written, good methodology and with appropriate references. Unless the use of competitive ELISA has been previously investigated for influenza A virus. There are multiple papers covering the competitive ELISA method for serodiagnosis of influenza A as doi: 10.4142/jvs.2009.10.4.323, doi.org/10.1186/s12985-021-01564-6,…

Please, clarify the main points in the present study ensuring and highlighting the novelty.

Author Response

Please, clarify the main points in the present study ensuring and highlighting the novelty.

We did an over all editing of the manuscript and tried to highlight the novelty. To our knowledge, our H5 cELISA the only assay that can detect antibodies to currently circulating 2.3.4.4b H5Nx viruses.

Reviewer 3 Report

Comments for the Author:

The study by Hochman O et al, titled “Development and validation of a recombinant-H5 2 hemagglutinin-based competitive ELISA for serodiagnosis of 3 influenza A virus H5 subtype antibodies”.  The manuscript is well written. However, I have one concern about this article.

Fig 1. Author should mark the H5 protein in the gel.

Author Response

Author should mark the H5 protein in the gel).

Locations for recombinant H5 proteins in the gel were described in the Figure 1 legend