Ganglioside Binding Assay: A Complementary Approach for Enhanced Tetanus Toxoid Quality Control

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors of this manuscript ("Ganglioside Binding Assay: A Complementary Approach for
Enhanced Tetanus Toxoid Quality Control) present a ganglioside binding assay as an alternative method to evaluate inactivation of tetanus toxin. A potential strength of the method presented is in possibly reducing animal testing. 

 

The authors also suggest the method may be an improvement over another alternative test (BINACLE) that has not been widely implemented due to certain technological limitations. However, the authors argue against what I strongly feel is an advantage of BINACLE over the method described in the manuscript - namely that BINACLE tests both ganglioside binding as well as toxin enzymatic activity (the assay described in the current manuscript does not evaluate enzymatic activity). I suggest the authors tone down this discussion (for example, lines 359-369, lines 395-404). The authors recognize the potential concerns and discuss work to develop a complementary enzymatic assay (406-414).

Author Response

Please see attached file.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript introduces a Ganglioside Binding (GB) assay as an in vitro alternative to reduce reliance on animal testing in the quality control of tetanus toxoids. The topic is relevant, timely, and aligned with the 3Rs principles and international regulatory discussions on vaccine testing. The correlation between the GB assay and Ramon’s flocculation method (R² = 0.999) and its concordance with guinea pig detoxification tests are notable findings. However, several critical weaknesses must be addressed before the manuscript can be considered for publication.

Introduction: while the background is informative and references some relevant work (e.g., BINACLE assay), the introduction lacks critical depth. The authors present the GB assay as novel but fail to adequately situate it in the context of prior in vitro alternatives (e.g., cell-based assays, FRET-based toxin detection, nanobioprobes). The claim of being the “first validated GB assay” is overstated, since ganglioside-based binding has been reported previously in toxin detection. The introduction should also better justify the added value of GB compared to BINACLE, beyond “simplification.”

Methodology: the methods are extensively described, but there are issues of reproducibility and transparency. The assay relies on proprietary vaccine manufacturer samples, which raises concerns about reproducibility and transferability to independent laboratories. For example, the ganglioside mixture source is described, but the composition and variability of bovine-derived gangliosides are not fully characterized, potentially limiting reproducibility. Moreover, the validation appears restricted to a single collaborating company (KM Biologics), which reduces generalizability. The use of guinea pig reversion tests is problematic, while the authors emphasize reduction of animal use, their validation still relies heavily on animal testing. A more robust demonstration would have included cross-validation with independent, fully in vitro methods.

Results: the data presentation is clear and figures are of good quality. Linearity and reproducibility are convincingly shown, but the biological significance is less clear. For instance: the assay detects “toxicity reversion” after heat treatment, but whether this reversion correlates with in vivo immunogenicity or long-term vaccine stability is not convincingly demonstrated. Statistical analyses are sometimes superficial, with excessive reliance on high R² values, which in practice may simply reflect careful calibration but not necessarily biological robustness.

Discussion:  is overly self-congratulatory and fails to critically address limitations. Important concerns remain unacknowledged: a).Will regulatory agencies accept the GB assay as a replacement, or only as a complementary tool? b).How does this assay compare to more comprehensive approaches like BINACLE in terms of sensitivity and regulatory readiness? c). Potential cross-reactivity with other clostridial toxins is not evaluated. d).The lack of discussion on scalability, inter-laboratory reproducibility, and regulatory acceptance significantly weakens the paper.

Conclusions: overstate novelty and regulatory impact. While the GB assay has potential as a complementary method, claiming that it can broadly replace animal testing is premature without multicenter validation.

 

Point-by-Point Evaluation

Does the introduction provide sufficient background and include all relevant references? Must be improved. Lacks critical context on previous ganglioside-based methods and alternative in vitro assays. Overstates novelty.

Is the research design appropriate? Can be improved. Validation limited to a single manufacturer and relies on animal testing, undermining the replacement claim.

Are the methods adequately described? Yes, but with caveats . Well detailed, but reproducibility outside the authors’ lab is questionable due to proprietary reagents.

Are the results clearly presented? Yes. Figures and tables are clear, although interpretation sometimes overstated.

Are the conclusions supported by the results? Can be improved.The results support GB as complementary, but not as a validated replacement. Conclusions exaggerate impact.

Are all figures and tables clear and well-presented? Yes. Graphs, tables, and SDS-PAGE data are presented clearly.

Originality/novelty? Average. Assay is incremental; binding-based assays already exist.

Significance of content? Average. Relevant for vaccine QC, but limited by single-lab validation.

Quality of presentation? High.  Well structured, logical, clear figures.

Scientific soundness? Average.  Data are solid but lack independent replication and broader validation.

Interest to readers? Average tohigh. Relevant to researchers in vaccine QC and toxin detection.

Overall Merit? Average. valuable as preliminary work, but not yet ready for definitive conclusions or regulatory impact.

Are the references cited in this manuscript appropriate and relevant to this research? Yes, but incomplete, additional references on alternative assays should be cited.

 

Recommendation: Major Revision (mot yet suitable for acceptance).

Although the GB assay is a promising complementary tool for tetanus toxoid quality control, the manuscript in its current form overstates novelty, lacks independent validation, and does not adequately discuss limitations or regulatory implications. Without multicenter validation and broader comparative analyses, the claim that GB assay can replace or meaningfully reduce animal testing remains premature.

I recommend that the authors: a) Substantially revise the introduction to better contextualize the assay. b). Provide a more balanced discussion, including limitations and regulatory challenges. c).Clarify how the GB assay compares to existing alternatives beyond BINACLE. d). Include additional validation with independent laboratories or complementary in vitro assays. 

Only after such revisions could the manuscript be considered for publication in Toxins.

Best regards, 

 

Author Response

Please see attached file.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I read the revised manuscript and the authors’ point-by-point cover letter. Overall, the paper is substantially improved (clearer positioning as a complementary, single-site validation; better discussion of limitations and regulatory pathway), but several issues remain notably residual overstatement and language/consistency problems in the manuscript text (despite the more balanced cover letter). I therefore recommend another revision before acceptance. Recommendation: major revision (textual/interpretive) no new animal work or multicenter data required for this round, but the manuscript text still needs (i) removal of remaining over-claims and inconsistencies, (ii) targeted language editing, and (iii) tighter alignment between abstract/key contribution and the toned-down claims in the Discussion/Conclusions presented in the cover letter.

What clearly improved: the authors now position the GB assay as a complementary screening tool and explicitly acknowledge single-laboratory scope and the need for multicenter validation and cross-reactivity studies; this is stated repeatedly in the responses and proposed revisions. They lay out a three-phase pathway (single-site → multicenter → regulatory adoption) and note cross-reactivity testing has been initiated. Methods are well specified (plates, coating, readout, instrument settings, validation steps), addressing reproducibility concerns at least within a site.  Figures/results convey linearity, specificity, stability, and toxin–toxoid discrimination, and the time-course/inactivation plus guinea-pig data show coherent trends. 

 
Remaining problems that block acceptance: residual overstatement & internal inconsistency (abstract/key contribution). The manuscript still contains over-claims inconsistent with the cover letter’s toned-down framing. The abstract/key contribution repeats strong language such as “first validated ganglioside binding assay,” “perfect correlation,” and “significantly reduces animal testing requirements,” which exceeds what a single-site proof-of-concept can support and contradicts the authors’ own revised Discussion stance.  In the cover letter, they retract such claims and reframe the work as foundational and complementary.  This mismatch must be corrected in the manuscript text itself (not just promised in the response).

Language quality and editorial issues in the manuscript. There are duplicated sentences and typos that undermine polish for example, abstract duplication (“This study introduces…”) and spacing/typographical errors, plus “remaied” in Figure 1 legend. 
 These require careful copy-editing.

Scope/claims still need tightening relative to data.The paper compellingly documents binding-based detection and shows concordance with animal outcomes in early inactivation phases, but it does not yet address cross-reactivity empirically nor inter-laboratory robustness acknowledged as future work in the responses.  The text should consistently present the assay as a screening alternative with future expansion (FRET/enzymatic readouts) rather than implying near-term replacement. 

Does the introduction provide sufficient background and include all relevant references? Can be improved. The authors added broader context and citations to alternative in-vitro methods (BINACLE, cell-based/FRET/nanobioprobes), improving balance; still, claims in the abstract/key contribution should be aligned with that framing. 

Is the research design appropriate? Can be improved. As a single-site validation tightly linked to a manufacturer’s materials, the design is appropriate for Phase 1 proof-of-concept, but generalizability remains limited until multicenter work is done; the text now acknowledges this. 

Are the methods adequately described? Yes. Reagent sources, plate prep, blocking, conjugate, readout parameters, calibration, and validation steps are delineated in sufficient detail for reproduction within a site. 

Are the results clearly presented? Yes. Dose–response discrimination (TeNT vs TeTd), linearity/specificity/stability, and time-course/reversion analyses are presented clearly with appropriate statistics and figure callouts. 

Are the conclusions supported by the results? Can be improved. The complementary, screening conclusion is justified; however, replacement-leaning phrasing and “first validated” claims in the abstract/key contribution are not warranted by single-site data. 

Are all figures and tables clear and well-presented? Yes. Figures sufficiently convey trends (e.g., survival curves, SDS-PAGE bands, linearity). Minor English edits to legends would help polish. 

Originality/novelty? Average. Binding to gangliosides is not new; the contribution is a streamlined, QC-oriented implementation with single-site validation and correlation, now properly framed as complementary. 

Significance of content? Average. Useful step toward 3Rs in QC workflows; significance will grow with multicenter evidence and cross-reactivity data (now planned). 

Quality of presentation? Average. Structure and data display are solid, but lingering abstract/Key Contribution overclaims and language slips reduce polish. 

Scientific soundness? Average. Assay analytics look robust (linearity, specificity, stability) and animal concordance is plausible; scope claims must match the evidence presented. 

Interest to readers? Average. Of interest to vaccine QC/regulatory science communities, especially those pursuing stepwise animal-use reduction.

Overall merit? Average. A credible Phase-1 in-vitro QC contribution publishable after one more revision focused on textual accuracy and consistency.

Actionable edits required for acceptance: purge remaining overstatements in Abstract and Key Contribution (e.g., remove “first validated,” “perfect correlation,” “significantly reduces animal testing requirements”); replace with the cover-letter’s foundational/complementary framing. Copy-edit to fix duplicated sentences, spacing, and typos (e.g., “remaied”). Align Conclusions to emphasize: (i) single-site scope; (ii) screening utility; (iii) planned multicenter/cross-reactivity/FRET enzymatic complement exactly as argued in the response but ensure this wording appears in the manuscript. 

Best regards,

Comments on the Quality of English Language

Quality of english language: the English could be improved to more clearly express the research. Resolve duplicated sentences and typographical errors noted above.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed the core concerns raised in the previous review by (i) substantially revising the Introduction to situate the ganglioside binding (GB) assay within the landscape of in-vitro alternatives and by tempering novelty claims; (ii) reframing the study as a single-laboratory, proof-of-concept validation rather than an immediate replacement for animal tests; and (iii) expanding the Discussion/Conclusions to acknowledge regulatory realities, study limitations, and an implementation pathway (single-lab → multicenter → regulatory consultation). They also added missing references on alternative methods and clarified the biological and statistical interpretation of their results. 

 

What was improved (with evidence from the response)

1) Context and novelty: the Introduction now cites prior ganglioside-based detection approaches and other in-vitro alternatives (including adding a new reference) and removes the “first validated GB assay” overstatement. This aligns the work as an incremental, simplified binding readout complementary to approaches like BINACLE. 

2). Scope and  claims: the Abstract, Discussion, and Conclusions explicitly present the study as Phase 1 single-site validation with strong correlation to traditional methods, not a comprehensive replacement. The regulatory acceptance is portrayed as contingent on future multicenter validation. 

3). Limitationsand  pathway: the authors add a clear limitations section (single manufacturer, absence of cross-reactivity data, need for inter-lab reproducibility) and outline a three-phase pathway toward potential regulatory use. 

4). Reproducibility details: they point to existing data (e.g., Figure 2F) showing lot-to-lot performance of the ganglioside mixture and discuss practicality versus purified components. 

5). Biological/statistical interpretation: They explicitly caution that a high R² reflects analytical agreement rather than comprehensive biological robustness or long-term stability/immunogenicity, which will need future studies. 

Remaining limitations (still acceptable if claims stay scoped)

1). Single-site validation: no new multicenter data were added; external transferability remains to be demonstrated. 

2). Cross-reactivity: testing against other clostridial toxins is only planned/in progress; no new results are included. 

3). Regulatory readiness: the method is correctly repositioned as a complementary screening tool; acceptance as a replacement is not claimed, but readers should not infer imminent replacement. 

 

Recommendation: Accept after minor revisions.

The revisions adequately address the conceptual oversell and provide the appropriate scientific framing for publication as a foundational, single-laboratory validation of a simplified GB assay with strong analytical correlation to established methods. Given the journal’s scope and the study’s contribution to the 3Rs agenda, publication is justified provided the manuscript consistently reflects the limited scope and clearly signals what remains to be done.

Required minor edits before acceptance

a) Consistency of scope across the manuscript: ensure every section (Title, Abstract, Graphical Abstract/Key Contribution, Conclusions) uniformly frames the work as a single-site proof-of-concept complementary assay, not a replacement. 

b). Limitations box (concise): add a short bullet list at the end of the Discussion summarizing: single-lab setting, lack of cross-reactivity results, and need for multicenter validation/technology transfer studies. 

c). Materials transparency: where feasible, provide catalog/source and specification ranges for the ganglioside mixture (e.g., composition class, purity range) and explicitly state any steps aiding tech transfer (e.g., acceptable alternatives if the specific mixture is unavailable). This strengthens reproducibility without disclosing proprietary information. 

d). Regulatory language: keep statements about regulatory use in conditional/future tense and tie them explicitly to successful multicenter validation and agency consultation. 

e). Data availability/transferability note: Add a brief statement on what information, SOPs, and acceptance criteria can be shared with external labs for planned inter-laboratory studies. 

If the authors implement the minor textual/structural edits above, I consider the manuscript suitable for publication as a methods-focused, proof-of-concept contribution that advances practical 3Rs-oriented QC workflows while accurately delineating its current evidence boundaries. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf