Development and Optimization of an Indirect Sandwich ELISA for Detection of Foot-And-Mouth Disease Virus Serotype O

Reviewer Comment

Response to Reviewer

What vaccine was used to inoculate the guinea pigs and rabbits? Is this from a virus that matches the O virus circulating in that region? If not, how would the sensitivity be increased compared to commercial tests? Vaccine details are needed.

We thank the reviewer for this important comment. Guinea pigs and rabbits were immunized using a commercially available oil-adjuvanted inactivated Foot-and-Mouth Disease Virus (FMDV) serotype O vaccine (UVAC-FMD®). This vaccine is formulated based on regionally relevant circulating strains and is widely used in Pakistan for vaccination programs. The use of such locally relevant antigenic material likely enhances antigen–antibody affinity and contributes to improved assay performance.

What was the purpose of the neutralization capacity?

We appreciate the reviewer’s query. The virus neutralization assay was performed to confirm the functional activity and specificity of the generated antibodies prior to their use in ELISA development.

Line 175 says serotype O antigen, was this an antigen or the whole virus? please provide detail

We thank the reviewer for pointing out this ambiguity. The antigen used in the assay was whole inactivated FMDV serotype O virus propagated in BHK-21 cell culture, not a purified subunit antigen. The manuscript has been revised for clarity.

Why did they try U-shape cell culture plates?

We appreciate this observation. U-shaped cell culture plates were included as part of a comparative assessment to evaluate the impact of plate geometry and surface properties on antigen–antibody interactions and assay performance. This allowed confirmation that high-binding ELISA plates provide superior performance.

Table 1: Were these the values of pooled serum samples (n=5)? There must have been individual variation if measured separately.

We thank the reviewer for this valuable comment. The values presented in Table 1 represent pooled serum samples (n = 5) within each group. Pooling was performed to obtain sufficient volume and reduce individual variability for assay standardization.

If the results show little difference between regular ELISA plate and Maxisorp Nunc, why choose the more expensive Maxisorp plate?

We thank the reviewer for this important point. Although comparable OD values were observed, Maxisorp high-binding plates were selected due to their superior protein-binding capacity, coating consistency, and reproducibility, which are critical for assay standardization and diagnostic reliability. These advantages may not be fully reflected by OD values alone.

Why aren't the performance results presented? How did this test compare to a commercial test?

We thank the reviewer for this important observation. The primary objective of this study was development and analytical optimization of the IS-ELISA under controlled laboratory conditions. A direct comparison with commercial kits and full diagnostic validation (sensitivity, specificity, ROC analysis) was beyond the scope of this study. We have revised the manuscript to avoid overstatement and clarified this limitation.

Reviewer Comment

Response to Reviewer

1. The study demonstrates that IEGPS antibodies improved neutralizing titres (1:128), however, low protein yield after ion exchange suggests significant loss during purification or high dilution, which may not be feasible for commercialization.

We appreciate this important comment. The purpose of ion-exchange purification in the present study was to enrich functionally relevant immunoglobulins and improve assay performance during laboratory standardization, rather than to establish an immediately commercial-scale production pipeline. Although the total protein yield decreased after purification, the purified IEGPS fraction retained functional neutralizing activity and performed better as a capture antibody in the optimized assay. We agree that purification yield, scale-up efficiency, and batch production feasibility are important considerations for commercialization and have now acknowledged this as a limitation of the study.

2. Achieving a detection limit of 10^1.2 TCID50/ml suggests the assay is competitive with several existing commercial platforms. However, simply stating it works similarly is insufficient. A formal statistical correlation (e.g., Cohen’s Kappa or ROC curve analysis) against a globally recognized gold standard kit must be included.

We thank the reviewer for this valuable suggestion. We agree that formal statistical comparison with a recognized commercial reference kit would substantially strengthen the manuscript. However, such comparative validation was not performed in the current study. Therefore, we have revised the manuscript to avoid overstating diagnostic performance and now describe the present work as analytical optimization and preliminary laboratory evaluation rather than full diagnostic validation. We have also added this as a limitation and identified direct comparison with commercial kits, ROC analysis, and agreement statistics as priorities for future work. Importantly, if the detection limit of 10^1.2 TCID50/mL is to be retained in the manuscript, it must be explicitly supported in the Results and Methods; otherwise it should be removed.

3. Sandwich and/or competition ELISAs for FMDV are already the gold standard. The manuscript does not introduce a new technology. It is essentially a local standardization paper. The narrative needs to shift from emphasizing that an ELISA was developed to why this ELISA is a superior or necessary alternative to existing global standards.

We agree with the reviewer. The manuscript has been revised to clarify that this work represents a local assay standardization and optimization study, not the introduction of a new immunodiagnostic format. The revised narrative now emphasizes the potential relevance of such an assay in settings where access to imported kits may be limited by cost, availability, or dependence on non-local reagents.

4. A neutralizing titre of 1:128 for the capture antibody is relatively low for a “high-sensitivity” claim. High-quality reference ELISAs typically utilize hyperimmune sera with titres >1:1000.

We thank the reviewer for this important point. We agree that the neutralizing titre of 1:128 alone may not justify a broad “high-sensitivity” claim when compared with reference-grade reagents used in established commercial or reference assays. In the revised manuscript, we have moderated this wording and now describe the assay as showing promising analytical performance under optimized laboratory conditions. Our interpretation is based on the observed assay discrimination and optimized signal-to-background ratio, rather than solely on the antibody neutralization titre.

5. For a diagnostic kit to be useful in the field, its shelf life and thermal stability must be documented.

We appreciate the reviewer’s comment and agree that reagent stability and shelf-life evaluation are essential for translation into a field-deployable diagnostic kit. However, such stability studies were not included in the present work, which was limited to assay development and laboratory optimization. We have now acknowledged this as an important limitation and future research priority.

6. How was the cut-off value determined? Was it Mean + 2SD or Mean + 3SD of negative controls? This is critical for the sensitivity calculation.

We thank the reviewer for highlighting this critical point. The manuscript requires clearer reporting of cut-off determination. If a cut-off was calculated during the study, the exact formula and data source should be explicitly stated in the Methods and applied consistently in the Results. If no formal cut-off analysis was performed, then sensitivity/specificity claims should be removed or revised, because they cannot be validly supported without a defined threshold.

Conclusion: suitable for publication, but not in a high-impact global immunology journal unless the authors can highlight a specific edge (e.g., lower cost or higher sensitivity than market leader).

We thank the reviewer for this constructive conclusion. We agree that the manuscript is better positioned as a regional diagnostic optimization and standardization study rather than a high-impact global technology paper. In response, we have revised the manuscript to better align its claims with the data generated and to emphasize its potential utility in endemic and resource-constrained settings. We have avoided unsupported superiority claims and clearly outlined the additional studies required for commercial and international benchmarking.

Reviewer Comment

Response

Manuscript Changes

1. Title claims “high sensitivity”; lack of PFU/LOD data, serial dilutions, and comparison with regional strains and commercial kits

We thank the reviewer for this important observation. We agree that the term “high sensitivity” requires quantitative validation. The current study focuses on assay optimization rather than full diagnostic validation. PFU/TCID₅₀-based limit of detection (LOD) and formal sensitivity assessment were not performed, and this has now been explicitly acknowledged. Claims regarding improved detection of regional strains have been moderated, and we clarify that comparative evaluation with field isolates and commercial ELISA kits will be conducted in future studies.

✔ Title revised (removed/toned down “high sensitivity”)

✔ Abstract and Conclusion moderated

✔ Limitation added in Discussion regarding lack of LOD and comparative validation

2. Neutralizing antibodies are not required for detection—why perform neutralization assay?

We agree that neutralizing activity is not essential for ELISA-based detection. The virus neutralization assay was performed to confirm the specificity, functional integrity, and biological activity of the generated hyperimmune sera prior to their use in assay development. This ensured that antibodies were immunologically relevant and structurally intact after purification. The purpose has now been clearly clarified in the manuscript.

✔ Clarification added in Materials & Methods (VNT section)

✔ Explanatory paragraph added in Discussion

3. No sensitivity/specificity determined; no comparison with gold standard; cost claim questionable due to serum use

We fully agree that diagnostic sensitivity, specificity, and comparison with gold-standard assay are essential. The manuscript has been revised to clearly state that this study represents laboratory-scale optimization, and that comprehensive diagnostic validation (including ROC analysis, sensitivity/specificity, and comparison with commercial ELISA kits) is required in future work. Regarding cost, we acknowledge that large-scale use of hyperimmune sera may increase cost and limit scalability. The claim of “low-cost” has been moderated, and future improvements such as monoclonal or recombinant antibody production have been proposed.

✔ Limitation section strengthened in Discussion

✔ Cost claim revised throughout manuscript
✔ Future work (ROC, Kappa, commercial comparison) added