An ELISA-Based Alternative to Mouse Bioassays for Quantitative Evaluation of Tetanus Toxin

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents a well-structured ELISA–IC system as a potential alternative to mouse bioassays for tetanus toxin evaluation. The analytical validation of the ELISA platform is rigorous, and the correlation with mouse MLD/LD₅₀ values across 18 isolates. However, a critical conceptual issue requires deeper discussion and clarification: the fundamental distinction between antigen quantification and functional bioactivity, which is a huge obstacle for this method to transfer to practical applications.

1. Please provide the origin of used antibody in this work in detail.
2. The ELISA developed in this study measures total immunoreactive tetanus toxin protein, whereas the mouse LD₅₀ assay evaluates functional neurotoxic activity in vivo. These two parameters are biologically related but not equivalent. The reported discrepancy range (0.71–6.03-fold variation between ELISA-derived values and mouse LD₅₀) highlights this intrinsic difference. Further mechanistic bridging between antigen quantity and in vivo bioactivity will be essential for practical applications?
3. Line 201, this method stands the highest sensitivity of reported method. Please provide relevant information to confirm it, otherwise, revise this statement.
4. The 18 bacterial strains originated from a single geographical location, and the data set is still relatively small, which may limit the significance of this work.
5. Additional discussion on the biochemical states of toxin (active vs. partially degraded vs. conformationally altered forms) and how these affect immunoreactivity versus biological lethality would significantly strengthen the scientific rigor of the study.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Authors describe an immunoassay development with the aim for measuring the presence of tetanus toxin directly from clinical or environmental samples that would be comparable to replace animal dependent assays. Astonishingly, such an assay does not yet exist. Overall, the methods, results and conclusions on the relevance of the study need improvement to deliver the message concisely.

Abstract:

• Define that 3R principles are related to ethical animal testing.
• Briefly describe what TT evaluation includes: proof of detoxification or activity, strain acitivity or quantity. What are the intended uses?
• Give an exact value for CV, intra or inter assay CV?
• Cross reactivity with what?
• I think R² and p-value are unnecessary for comparison of ELISA and lethal dose comparison, the (pearson?) correlation is impressive enough alone. (Same applies for results section)
• Instead of " Both ELISA quantification methods", describe the two for clarity.
• I think the term “ELISA-IC” is misleading or easily misinterpreted, use ELISA and IC systems/methods/platforms

 

Introduction:

• The research on finding alternative ways for measuring TT and DT potency is vast. The narrative of the introduction and manuscript should be further aligned with previous efforts: how does this assay compare with other efforts of ELISA, TOBI, Vero Cell assays, BINACLE techniques intended for vaccination validation. Why can’t they be used for clinical or environmental testing? Why is there a need for a diagnostics TT-test? Please describe the typical diagnosis alternatives in clinical case – what it the typical diagnostic pathway and techniques used for diagnosing tetanus infection? What is the time frame that a patient need care? Is there time to isolate the pathogen, cultivate it, and test with ELISA? Is quantitative readout needed and why? What are the advantages for the developed test? The IC test is not quantitative (consider fluorescent labels or absorbance intensity meters in future?), what are the limitations? This should be introduced or discussed extensively.
• The used references to introduce the subject are rather old. E.g., referencing limitation on the studies on comparing antigen and biological activity (ref 16) from 2006, or then confirm, that no new reviews or efforts on the matter exist.

 

M&M:

• Tt stock concentration is not relevant information. Is TT-001 toxoid or toxin? Is it the same protein as used for rabbit immunization? IF not, please specify the source of different proteins or the detoxification methods.
• Line 331: "Samples were stored at -80" - What samples - the strains? Or does this replicate line 337 sentence? Please remove if duplicated info.
• Lines 331-335 replicates twice the previous study idea, please reformulate the very long sentence.
• How was the conjugation done to HRP (very standard yes, but briefly explained or cited)?
• What was the source of toxins from other clotridium species? Or cultured similarly as in 5.1?
• IC details: please provide producers of strip materials (also backing plastic?), and nitrocellulose pore sizes, how much gold label was used per strip, how much antibody capture was used - how was the polyclonal antibody dispensed as a test line? What is the control line (Based on Figure 5 there is one)? What amount of protein is there in control? The ELISA used 50µl of sample, and IC 100µls = were the samples further diluted or was the amount of analyte just doubled?
• Please provide software version (if applicable) for Bioassay Assist statistical analysis software (NIID).
• Reference 18 appears as the last reference in M&M, please check the order of citations and references. There is no citation to reference 17, citation 19 is also misplaced (comes after 25).

 

Results:

• Based on methods or results it is not evident how many replicates were measured to evaluate CV (24 of each in figure 1b? or 8+8+8?). How many replicas for 1A?
• Based on the methods or results it is not clear how LoQ has been defined and calculated, what is the blank reference sample, how many replicas were used for blank sample.
• Figure 4 lacks bolded first line (inconsistent with other Figures).
• Please explain what ”Error bars represent ±1 standard deviation.” exactly means? Based on variation between replicas or artificial assuming standard distribution?
• Table 1: what are the acceptance criteria based on? Please give a reference. If self-defined, they don’t provide much value. Although Table 1 nicely gathers the findings, it replicates other figures and main text, and could be considered as a presentation of duplicate results unnecessarily.
• Table 2 would be easier to interpret if the data were sorted based on ELISA relative values from smallest to largest. Table 5, similar to table 2. Table 6 similar to 2.
• Text lines 121-123 fully replicates info from Table 3: I would suggest removing most of the text and simply citing the table. Line 124-5 is not necessarily a descriptive result of this study, consider moving to discussion to give rationale for the need of quantitative assays.
• Table 3 legend seems wrong since there are no comparisons to ELISA.
• Figure 3 (and 4) bacterial species in italics.
• Figure 3a shows only 9 dots, when 10 samples are described. Figure 4 only shows 15 dots out of 18: consider removing the fill from the markers (only borders colored) to distinguish overlapping datapoints.
• Please confirm how Figure 3 and Table 2 differ from each other? If the data is duplicate, the other format should be removed.
• Figure 3 explains the color coding twice, confirm from journal editorial guidelines whether the labels are to be shown only as text, and preferably remove the ones in the illustrations.
• Please define Figure 3a x-axis and 3b x and y axis in the figure more accurately what parameters they describe, tetanus toxin concentration? (3b LD50).
• I am not certain, but does figure 3b replicate table 5 results (only difference is log transfer?)? Think of how to show the results without unnecessary duplication. Based on comments thus far, the number of Figures and tables could be cut considerably, making the manuscript easier (=faster) to grasp for the audience.
• Does Figure 4 combine data from Figure 3a and table 4? Once again, the data has been duplicated (if not triplicated already!). Preferably, combine results, or reduce time dependent narrative (first we did the environmental, then clinical…) to justify presenting them at the same time. I would suggest that moving Figure 4 to replace 3a would do it.

 

Discussion:

• Line 191-2 describing results is unnecessary. Similarly lines 208-226 is results heavy, remove numbers and focus on conclusions.
• “which compares favorably with previously reported methods for detecting tetanus toxin.” Please provide references.
• How do the authors think “rapid” on-site testing with ICs is possible for Clostridium cultures? Consider the narrative of discussion accordingly, if this obvious limitation for sample handling exists.
• Please add a chapter discussing study limitations.
• Please expand rationale how ELISA-IC combination would be worthwhile? What is the added value to get a quantitative results after semi-quantitative IC?
• Polyclonal capture antibodies: stock differences, mab development, or necessity for strain variation to have pabs? Monoclonal would further reduce the risk of cross-reactivity to other Clostridium species?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have covered all previous comments critically. With revision, comes typos, which I encourage to examine upon pre-proofing: e.g., p12 "Overall, 18 representative C. tetani strains with diverse toxin-production capabilities were for this study."