Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents the development of an indirect ELISA to detect human IgGs in the serum for the Nipah virus glycoprotein. A commercial recombinant protein is used as antigen and serum samples (positive or negative) are reference sera or are previously tested. Qualification studies were performed for the indirect ELISA developed by the authors and these studies were the focus of the manuscript.

The optimization of the indirect ELISA protocol is mentioned in the “Abstract” section, however details and results were not presented in the manuscript or commented in the “Result” section (Which assay step was optimized?).

For most parameters evaluated in qualification studies, results are analyzed and summarized in tables without representative graphs of the assay.

The manuscript purpose is the qualification of the indirect ELISA and relevant results should be presented to support the results. Graphs showing ELISA results are crucial for the assay qualification and should be considered.

“Introduction” section is concise and short. Previous assays developed to detect Nipah virus are not mentioned to show the novelty is not evident. In “Discussion” section, previous works are cited, however they were partially discussed and  compared with the present works.

In “Materials and Methods” section, specify the types of samples tested for each qualification study and the dilution factor used.

Fig. 2 y-axis: It is indicated as “Response (nm)”, but it refers to the absorbance reading at 450 nm.

Results of qualification studies are presented in incomplete way and it is recommended reviewing the manuscript. The results can be added as Supplementary material if the authors prefer.

The assay validation of this indirect ELISA will be reached by applying the assay to real samples for the screening patients with suspicion of Nipah virus infection or by evaluating the vaccine performance.

Author Response

Authors Response to Reviewer-1 Comments

 

Comments and Suggestions for Authors

The manuscript presents the development of an indirect ELISA to detect human IgGs in the serum for the Nipah virus glycoprotein. A commercial recombinant protein is used as antigen and serum samples (positive or negative) are reference sera or are previously tested. Qualification studies were performed for the indirect ELISA developed by the authors and these studies were the focus of the manuscript.

 

Comment: The optimization of the indirect ELISA protocol is mentioned in the "Abstract" section, however details and results were not presented in the manuscript or commented in the "Result" section (Which assay step was optimized?).

 

Authors' Response: We thank the reviewer for highlighting this point. We have now explicitly described the ELISA optimization steps in the manuscript.

In Section 2.3 Optimization of ELISA, under Materials and Methods, describe the optimization steps:

• Coating antigen concentration (0.2–3 µg/mL) to determine optimal antigen density.
• Serum dilution factor (1:24) based on CDC heat-inactivation guidance.
• Secondary antibody dilution (1:5000) to achieve optimal signal-to-noise.
• Substrate incubation time (10, 15, 20 min; 15 min optimal).
• Blocking buffer (1% BSA vs 5% skim milk; 1% BSA selected).

The corresponding results and heatmap are provided in Section 3 (Results; Optimization of Anti-Nipah IgG ELISA), Figure 1, which illustrates the effect of each parameter on optical density and overall assay performance.

 

Comment: For most parameters evaluated in qualification studies, results are analyzed and summarized in tables without representative graphs of the assay.

 

Authors' Response: Thank you for the comment. We will share all the figures as a supplementary file, which includes representative graphs of the assay.

 

Comment: The manuscript purpose is the qualification of the indirect ELISA and relevant results should be presented to support the results. Graphs showing ELISA results are crucial for the assay qualification and should be considered

 

Authors' Response: Thank you for the comment. We will share all the figures as a supplementary file, which includes representative graphs of the assay.

 

Comment:"Introduction" section is concise and short. Previous assays developed to detect Nipah virus are not mentioned to show the novelty is not evident. In "Discussion" section, previous works are cited, however they were partially discussed and compared with the present works. In "Materials and Methods" section, specify the types of samples tested for each qualification study and the dilution factor used.

 

Authors' Response: Thank you for the comments. Please find our point-by-point response below:

• Introduction (Section 1): We have expanded the background to include previously developed Nipah virus serological assays, highlighting their methodologies, advantages, and limitations. This now clarifies the novelty of the current BSL-2-compatible, quantitative ELISA targeting the G glycoprotein.
• Discussion (Section 4) We have enhanced the comparative discussion, explicitly highlighting differences in assay design, biosafety requirements, sample handling, and analytical performance relative to prior works.
• Materials and Methods (Section 2.3–2.6) We now explicitly indicate the sample types used for each qualification parameter: reference sera (NV-1, NV-2, NV-4, NV-6, NV-10), archived positive samples (NHP-1 to NHP-7), and negative controls (NC-1 to NC-8, CNC). All samples were prediluted 1:24 before testing. These details are reflected in Table 1 (sample summary). The results of the qualification studies are shown in Tables 2–6 (sensitivity, specificity, precision, accuracy, linearity, and detection/quantification limits) and Figures 1–3 (heatmap, linearity, and detection/quantification limits).

 

 

Comment: Fig. 2 y-axis: It is indicated as "Response (nm)", but it refers to the absorbance reading at 450 nm.

 

Authors' Response: We have corrected the y-axis label in Figure 2 of the manuscript to "Absorbance at 450 nm (OD units)" to reflect the measurement accurately.

 

Comments: Results of qualification studies are presented in incomplete way, and it is recommended to review the manuscript. The results can be added as Supplementary material if the authors prefer

 

Authors' Response: To provide a comprehensive visualization of the qualification studies, all graphical data supporting optimization, linearity, and detection/quantification limits can be included in a drive link, where you will find representative results.

 

Comments: The assay validation of this indirect ELISA will be reached by applying the assay to real samples for the screening patients with suspicion of Nipah virus infection or by evaluating the vaccine performance.

 

Authors' Response: We again thank the reviewer for highlighting the need for further validation. We have now explicitly acknowledged in the Discussion (Section 4) that full validation of the assay will require testing on larger panels of positive and negative sera, samples from diverse geographic regions, longitudinal samples from convalescent individuals, and specimens from vaccinated participants once available. We also note that field validation in endemic settings, inter-laboratory comparability using the WHO/NIBSC International Standard, and correlation of ELISA-derived IgG concentrations with neutralizing antibody titers will be essential to assess real-world assay performance, reproducibility, and operational feasibility

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for the opportunity to review the manuscript entitled Development and Qualification of a Nipah virus Glycoprotein-specific IgG ELISA for the Assessment of Human Antibody responses by Alam et al.

The manuscript describes the development of an ELISA with glycoprotein G coated plates and validation with clinical serum samples.

Major comments and concerns:

Not all methods are described clearly, for example

In the materials and methods section there seems to be some confusion over the unitage and description of the International Standard. Line 81 does not sufficiently describe the standard used and should reference NIBSC code 22/130_BA as the WHO ECBS endorsed two standards for harmonisation of either neutralisation assays or binding assays. Line 82 also incorrectly states that there is 1000 IU per ampoule, the instructions for use state that there is 250 IU/ampoule. This brings into doubt the calculations performed for the LLOD and LLOQ cited as it is not clear that the correct IU/mL was used for the calculations. Table 1 does correctly state 1000 IU/mL for NV-1.

The authors state the assay is calibrated to the IS however in the methods there is no indication on how this is done it only states “For assay qualification, sample concentrations were calculated in international units per millilitre (IU/mL) using SoftMax Pro software (version 136 7.1.1, GxP edition, Molecular Devices).” There is no evidence of how may points were performed if using parallel line analysis or if single point calibration for samples. Was the IS run on every plate or did they calibrate a secondary standard to the IS and then use this for relative potency calculations. The authors should add more information to the materials and methods to describe how this was done.

The recombinant glycoprotein G from Creative Diagnostics, used to coat the plates, should be further described, is it sequence relating to Bangladesh or Malaysia isolates and what is the potential impact on the ELISA sensitivity.

The referencing is not accurate in some places and mis-cites in others, the referencing requires reviewing.

The authors should comment in the discussion on the limit of the study, for example, a full validation is not possible due to limited sample availability. 

Authors have indicated 2 references where ELISA have been developed and validate for Nipah protein therefore provide a commentary as to the novelty of this work.

Monir comments:

Line 28 and 68: it should be only: WHO International Standard for antibodies (NIBSC is the custodian lab)

Line 80 what's n.d.-a?  they should reference the WHO website WHO/BS/2023.2458 WHO 1st International Standard for Nipah virus antibody

 

Line 81:  The Standard was not name NV1 but that was the code for the collaborative study. Use the NIBSC code 22/130 and two standards have been produced 22/130_NT and 22/130_BA. Recommend rewritten as follows: The WHO IS for anti-Nipah Virus Antibodies for binding assays (22_130_BA) was established with an assigned potency of 250 IU per ampoule.

Line 82:  the collaborative study samples are used as samples with a known titre, based on the consensus from the collaborative study results, rather than "reference sera".  Please modify the sentence as this is misleading.

Table 1: line 91 should be consensus value rather than reference value? Please describe how the confidence interval calculated?

Line 97: I believe only the sample 1-5 were provided from MHRA, please describe the origin of other samples and also provide the ethical statement for their donation and usage

Line 128-131: it is unclear whether the cut off is determined for each plate as 2x the average of blank OD + SD or as per line below 0.1

line 139:  Why did the author used ICH Q2(R1) rather than the revised Q2(R2) version? The reference [16] is author's previous work on development of a Nipah neutralisation assay. The correct reference for this sentence should be 19 or 20 (there are the same from different sources).

Line 146 same as line 139. Inappropriate use of auto citation.

Line 158 same as line 139. Inappropriate use of auto citation.

Line 154: the reference for the definition of CV(%) should not be a manuscript on a qRT-PCR. A reference is actually not needed as this is quite well established. Although please correct the formula as there is no % after 100 

Line 148: accuracy of the assay is distinct from Precision (as explained in line 167). Please change the wording.

Line 152: the A in ELISA stands for Assay, so tow word assay is therefore redundant

The ICH Q2(R1) guidelines do not actually state the acceptable range of R2 for linearity. References 22 and 23 do not look at linearity.   The choice of 0.8 and 1.25 should be justify in a different manner or relevant references provided.

Line 175: unclear use of the reference 18 to support a definition included in the ICH guidelines, ref 19 not 16

Line 191-2: Please describe how the confidence intervals (CIs) were calculated

Figure 1. Please plot individual lines as well as the merged data, in the graph it is not possible to distinguish the 3 samples. It will be important to see that results of each sample dilution as well as the overall result. Why only 11 points instead of 12? The figure legend it should be indicated what the 2 red lines mean.

Line 278: avoid using NV-1 as this is misleading, once the WHO IS is established the code number or the full name should be used (see comment in relation to Line 81).

Line 281: the citation 24 is not the ICH Q2(R2) but the M10, and why mentioning the R2 now, after using R1 for the entire manuscript?

 

Comments on the Quality of English Language

The consistency of language could be improved, for example citing materials consistency.

Line 83: included should this be "conducted"?

Line 84: were from Nipah survivors

Line 101: the paragraph title is misleading: it does not describe an optimisation, just the method- please correct

Line 119: “the HRP”, please change to “HRP-conjugated secondary antibody”

Line 162: Please rephrase: "Preferably the straight line should be straight".. suggestion:  the value obtained for each dilution point should be close to the regression line, with a correlation coefficient close to 1.

Line 180: Suggest substitute word "standards" with "criteria" or "specification" and "validation" with "qualification"

Line 181: Suggest substitute "acceptable" with "target"

Line 202: the spacing is wrong, please amend “withi n t he accept able”

 

Author Response

Authors Response to Reviewer-2 Comments

 

Reviewers' Comments and Suggestions for Authors

Thank you for the opportunity to review the manuscript entitled Development and Qualification of a Nipah virus Glycoprotein-specific IgG ELISA for the Assessment of Human Antibody responses by Alam et al.

The manuscript describes the development of an ELISA with glycoprotein G coated plates and validation with clinical serum samples.

 

Major comments and concerns:

 

Comments: Not all methods are described clearly, for example. In the materials and methods section there seems to be some confusion over the unitage and description of the International Standard. Line 81 does not sufficiently describe the standard used and should reference NIBSC code 22/130_BA as the WHO ECBS endorsed two standards for harmonisation of either neutralisation assays or binding assays. Line 82 also incorrectly states that there is 1000 IU per ampoule, the instructions for use state that there is 250 IU/ampoule. This brings into doubt the calculations performed for the LLOD and LLOQ cited as it is not clear that the correct IU/mL was used for the calculations. Table 1 does correctly state 1000 IU/mL for NV-1.

 

Authors' Response: We thank the reviewer for highlighting this very crucial point. We acknowledge that our initial description was unclear. The WHO/NIBSC International Standard (NIBSC code 22/130_BA), which is specifically endorsed for binding assays, is labeled as NV-1, and all other reference sera were similarly labeled by the MHRA when provided to us. We have followed these labels exactly to avoid any confusion.

The ampoule contains 250 IU of anti-Nipah virus antibodies. Upon reconstitution of the freeze-dried serum with 0.25 mL of nuclease-free water, the resulting concentration becomes 1000 IU/mL, as reported in Table 1. The calculation is as follows:

 

This clarification has been added to Section 2.1. All calculations for LLOD and LLOQ were confirmed using this 1000 IU/mL value, ensuring that the reported results are accurate and consistent with the MHRA-provided reference labels.

 

 

Comments: The authors state the assay is calibrated to the IS however in the methods there is no indication on how this is done it only states “For assay qualification, sample concentrations were calculated in international units per millilitre (IU/mL) using SoftMax Pro software (version 136 7.1.1, GxP edition, Molecular Devices).” There is no evidence of how may points were performed if using parallel line analysis or if single point calibration for samples. Was the IS run on every plate or did they calibrate a secondary standard to the IS and then use this for relative potency calculations. The authors should add more information to the materials and methods to describe how this was done.

 

Authors' Response: We thank the reviewer for highlighting this important point. We have now clarified in Section 2.5 how the assay was calibrated to the WHO/NIBSC International Standard (NV-1, NIBSC code 22/130_BA). The calibration was performed using a parallel-line approach as follows:

• NV-1 was prediluted at 1:24, followed by two-fold serial dilutions across 8 wells (rows A–H) on each plate.
• Sample sera were diluted in parallel on the same plate.
• Dose-response curves of both the reference and the samples were fitted using SoftMax Pro software (version 7.1.1, GxP edition).
• Sample concentrations were then calculated in IU/mL relative to NV-1.

This procedure ensures that all reported concentrations, including LLOD, LLOQ, and other quantitative qualification parameters, are fully traceable to the International Standard.

This explanation has been added to the manuscript to provide complete transparency of the calibration procedure and to confirm that all quantitative results are accurately expressed relative to the WHO/NIBSC standard.

 

Comments: The recombinant glycoprotein G from Creative Diagnostics, used to coat the plates, should be further described, is it sequence relating to Bangladesh or Malaysia isolates and what is the potential impact on the ELISA sensitivity.

 

Authors' Response: We thank the reviewer for this question. Published sequence analyses indicate that the Nipah virus glycoprotein G is highly conserved between Bangladesh and Malaysia strains, with approximately 95.5% amino acid identity reported in comparative analyses of full‑length G sequences. Such high sequence similarity, including conserved structural domains, suggests that the recombinant G protein used in our ELISA is likely to be recognized by antibodies generated against either strain, supporting broad assay sensitivity. This has been added to the discussion section 4.

 

Comments: The referencing is not accurate in some places and mis-cites in others, the referencing requires reviewing.

 

Authors' Response: Thank you for the comment. We have made corrections in the revised manuscript.

 

Comments: The authors should comment in the discussion on the limit of the study, for example, a full validation is not possible due to limited sample availability.

 

Authors' Response: We thank the reviewer for this comment. We have now explicitly detailed the study’s limitations in the discussion section of the manuscript. Specifically, we note that a full validation of the ELISA across a broader range of clinical and vaccinated samples was not possible due to limited sample availability. While the current qualification demonstrates strong analytical performance, the limited sample panel restricts generalization across diverse populations and geographic regions. Future studies, including larger panels of positive and negative sera, longitudinal samples, and specimens from vaccinated participants, will be necessary to confirm the assay’s performance and applicability in real-world and field settings.

 

Comments: Authors have indicated 2 references where ELISA have been developed and validated for Nipah protein therefore provide a commentary as to the novelty of this work.

 

Authors' Response: We thank the reviewer for the comment. In previous ELISAs for Nipah virus G protein, several contributions have been described [7,8]. Our work provides several novel contributions. First, the assay is calibrated to the WHO/NIBSC International Standard, allowing standardized reporting in IU/mL and enabling cross-laboratory comparison. However, in the past, all ELISAs were developed as qualitative (Spot ELISA); in our case, we developed a quantitative ELISA that allows us to determine IgG values in IU (International Units). Second, it employs a recombinant G protein in a BSL-2-compatible workflow, reducing biosafety requirements compared with live-virus assays. Third, we have developed an in-house ELISA that can be easily performed with commercially available reagents. Our method is open to development in any research facility and is also reproducible. However, in previous publications/studies, we found that the ELISA was developed using in-house NiV antigens. Fourth, we have applied parallel-line calibration and rigorous analytical qualification, and have reported detailed metrics for sensitivity, specificity, linearity, precision, and detection limits. Finally, the assay is designed for practical applications in serosurveillance, outbreak investigations, and vaccine immunogenicity studies, providing a scalable and reproducible platform. These features collectively distinguish our work from previously published articles.

 

 

Minor comments:

 

Comments: Line 28 and 68: it should be only: WHO International Standard for antibodies (NIBSC is the custodian lab).

 

Authors' Response: Please find the revisions in the revised and updated manuscript file on page 1, lines 28-30.

 

Comments: Line 80 what's n.d.-a? they should reference the WHO website WHO/BS/2023.2458 WHO 1st International Standard for Nipah virus antibody

 

Authors' Response: Please find the revisions in the revised and updated manuscript file.

 

 

Comments: Line 81: The Standard was not name NV1 but that was the code for the collaborative study. Use the NIBSC code 22/130 and two standards have been produced 22/130_NT and 22/130_BA. Recommend rewritten as follows: The WHO IS for anti-Nipah Virus Antibodies for binding assays (22_130_BA) was established with an assigned potency of 250 IU per ampoule.

 

Authors' Response: We thank the reviewer for highlighting this point. We acknowledge that our initial description was unclear. The WHO/NIBSC International Standard (NIBSC code 22/130_BA), which is specifically endorsed for binding assays, is labelled as NV-1, and all other reference sera were similarly labelled by the MHRA when provided to us. We have followed these labels exactly to avoid any confusion.

The ampoule contains 250 IU of anti-Nipah virus antibodies. Upon reconstitution of the freeze-dried serum with 0.25 mL of nuclease-free water, the resulting concentration becomes 1000 IU/mL, as reported in Table 1. The calculation is as follows:

 

This clarification has been added to Section 2.1. All calculations for LLOD and LLOQ were confirmed using this 1000 IU/mL value, ensuring that the reported results are accurate and consistent with the MHRA-provided reference labels

 

Comments: Line 82: the collaborative study samples are used as samples with a known titre, based on the consensus from the collaborative study results, rather than "reference sera". Please modify the sentence as this is misleading.

 

Authors' Response: Please find the revisions in the revised and updated manuscript file .

 

Comments: Table 1: line 91 should be consensus value rather than reference value? Please describe how the confidence interval calculated?

 

Authors' Response: We received the reference values from our donor, CEPI, and the details of the confidence interval were provided and updated in the revised manuscript on page 7, lines 279-283.

 

Comments: Line 97: I believe only the sample 1-5 were provided from MHRA, please describe the origin of other samples and provide the ethical statement for their donation and usage

 

Authors' Response: Please find the revisions in the revised and updated manuscript file, pages 4-5; lines 154-158.

 

Comments: Line 128-131: it is unclear whether the cut off is determined for each plate as 2x the average of blank OD + SD or as per line below 0.1

 

Authors' Response: Thank you, reviewer, for pointing out my mistake here. The cut-off should be determined for each plate as 2x the average of blank OD + SD, as we have shown in the manuscript. I have removed the confusion caused by (x≥0.1) on page 6, lines 238-240.

 

Comments: line 139: Why did the author used ICH Q2(R1) rather than the revised Q2(R2) version? The reference

 

Authors' Response: We thank the reviewer for this comment. There was an error while updating the manuscript. We have added and revised the ICH Q2(R2) and updated it in the manuscript file (reference).

 

Comments: [16] is author's previous work on development of a Nipah neutralisation assay. The correct reference for this sentence should be 19 or 20 (there are the same from different sources).

 

Authors' Response: Thank you, it has been addressed, and the reference has been updated

 

Comments: Line 146 same as line 139. Inappropriate use of auto citation.

 

Authors' Response: Thank you. It has been addressed. Please find the revisions in the revised and updated manuscript file.

 

Comments: Line 158 same as line 139. Inappropriate use of auto citation.

 

Authors' Response: Thank you. It has been addressed. Please find the revisions in the revised and updated manuscript file.

 

Comments: Line 154: the reference for the definition of CV(%) should not be a manuscript on a qRT-PCR. A reference is actually not needed as this is quite well established. Although please correct the formula as there is no % after 100

 

Authors' Response: Thank you. It has been addressed. Please find the revisions in the revised and updated manuscript file.

 

Comments: Line 148: accuracy of the assay is distinct from Precision (as explained in line 167). Please change the wording.

 

Authors' Response: Thank you. It has been addressed. Please find the revisions in the revised and updated manuscript file in sections 2.6.2 and 2.6.4.

 

Comments: Line 152: the A in ELISA stands for Assay, so tow word assay is therefore redundant

 

Authors' Response: Thank you. It has been addressed. Please find the revisions in the revised and updated manuscript file.

 

 

Comments: The ICH Q2(R1) guidelines do not actually state the acceptable range of R2 for linearity. References 22 and 23 do not look at linearity. The choice of 0.8 and 1.25 should be justify in a different manner or relevant references provided.

 

Authors' Response: Apologies for our typing mistake. We thank the reviewer for this comment. We have made the correction in the reference from ICH Q2(R1) to ICH Q2(R2) for linearity in the manuscript.

 

Comments: Line 175: unclear use of the reference 18 to support a definition included in the ICH guidelines, ref 19 not 16

 

Authors' Response: Apologies for our typing mistake. All the confusion related to ICH Q2(R2) guideline has been made in the manuscript.

 

Comments: Line 191-2: Please describe how the confidence intervals (CIs) were calculated

 

Authors' Response:  The details of the confidence interval were provided and updated in the revised manuscript on page 7, lines 279-283.

 

Comments: Figure 1. Please plot individual lines as well as the merged data, in the graph it is not possible to distinguish the 3 samples. It will be important to see that results of each sample dilution as well as the overall result. Why only 11 points instead of 12? The figure legend it should be indicated what the 2 red lines mean.

 

Authors' Response: This has been renumbered as Figure 2 in the updated manuscript, and graphs for all 3 samples have been provided. However, there are 11 points in total; we are a little confused about the 12 points. We have only 11 points in our manuscript. And figure legends have also been updated on page 12, lines 398-400.

 

Comments: Line 278: avoid using NV-1 as this is misleading, once the WHO IS is established the code number or the full name should be used (see comment in relation to Line 81).

 

Authors' Response: We thank the reviewer for highlighting this point. We acknowledge that our initial description was unclear. The WHO/NIBSC International Standard (NIBSC code 22/130_BA), which is specifically endorsed for binding assays, is labeled as NV-1, and all other reference sera were similarly labeled by the MHRA when provided to us. We have followed these labels exactly to avoid any confusion. We corrected the IS as NV-1 WHO IS in the revised manuscript.

 

Comments: Line 281: the citation 24 is not the ICH Q2(R2) but the M10, and why mentioning the R2 now, after using R1 for the entire manuscript?

 

Authors' Response: We are extremely sorry for all this confusion. All the confusion related to the ICH Q2(R2) guideline has been addressed in the revised manuscript and in the reference.

 

 

Comments on the Quality of English Language

 

Comments: The consistency of language could be improved, for example citing materials consistency. Line 83: included should this be "conducted"?

 

Authors' Response: Thank you for the comment, the references have been revised.

 

Comments: Line 84: were from Nipah survivors

 

Authors' Response: Thank you for the comment. It has been addressed

 

Comments: Line 101: the paragraph title is misleading: it does not describe an optimisation, just the method-please correct

 

Authors' Response: We have the detailed optimization steps in the revised manuscript on pages 5-6, lines 163-209.

 

Comments: Line 119: “the HRP”, please change to “HRP-conjugated secondary antibody”

 

Authors' Response: Thank you for the comment. It has been addressed on pages 6, lines 228-229

 

Comments: Line 162: Please rephrase: "Preferably the straight line should be straight".. suggestion: the value obtained for each dilution point should be close to the regression line, with a correlation coefficient close to 1.

 

Authors' Response: Thank you for the comment. It has been addressed on pages 7, lines 278-280.

 

Comments: Line 180: Suggest substitute word "standards" with "criteria" or "specification" and "validation" with "qualification"

 

Authors' Response: Thank you for the comment. It has been addressed.

 

Comments: Line 181: Suggest substitute "acceptable" with "target"

 

Authors' Response: Thank you for the comment. It has been addressed.

 

Comments: Line 202: the spacing is wrong, please amend “withi n t he accept able”

 

Authors' Response: Thank you for the comment. It has been addressed.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed adequately comments in the letter and the revised version of manuscript is clear and interesting.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed all comments raised at review and I consider the manuscript suitable for publication.