Development of Anti-Peptide Antibody Specific for IgM Heavy Chain of Oryzias latipes and Its Application to Assay of Immune Response Triggered by BSA-Coated Microplastics

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study by Kizhakkumpat et al. aimed to develop a highly reactive and specific anti-peptide antibody against the IgM heavy chain of the medaka fish (Oryzias latipes), using an antigen peptide corresponding to the region between the CH3-CH4 domains of IgM. Additionally, the study seeks to explore the potential of this antibody as a tool to detect both systemic and mucosal immune responses, evaluating the impact of microplastics as adjuvants in immersion immunizations. This highlights the importance of the study for sustainable aquaculture.

Overall, the study is relevant for publication in the international journal Journal of Marine Science and Engineering.

The introduction is well-written and highlights the most important aspects for understanding the study. The materials and methods are adequate, as are the results and discussion. The conclusions also support the main findings of the study.

I have a few recommendations. The main highlights are:

• Lines 163-165: What tests of normality and homogeneity were used?
• The authors report that they performed a t-test to check for significant differences between treatments; however, the t-test is only used to assess statistical differences when there are two experimental groups. The fish were exposed to five different experimental groups. Please carefully check this information.

Author Response

Comments 1: I have a few recommendations. The main highlights are:

• Lines 163-165: What tests of normality and homogeneity were used?
• The authors report that they performed a t-test to check for significant differences between treatments; however, the t-test is only used to assess statistical differences when there are two experimental groups. The fish were exposed to five different experimental groups. Please carefully check this information

Authors' reply: Thank you for the critical and valuable comments. The t-test was done after the test for normality and equal variation by F-test. However, more importantly, as the reviewer pointed out, the use of t-test alone was not suitable for the detection of significant difference between the two groups among the five. 
Accordingly, we have tested the significance of difference among the five groups using one-way ANOVA with post hoc  Tukey's Honest Significant Difference test. As a result, we detected significant difference of antibody titers between the groups shown in revised Fig. 10.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript titled :Development of anti-peptide antibody specific for IgM heavy chain of Oryzias latipes and its application to assay of immune response triggered by BSA-coated microplastics is very designed study with high quality data. The experiments were performed with triplicates to make sure the quality of the data. The references are up to date. There are a few points need to be addressed before publication.

Line 104: why the 10% polyacrylamide gels instead of 4-12% gradient polyacrylamide gels? What is the quantity of protein sample was loaded into the gel?

Line 121: the quantity of the sample?

Line 140: in this section, what is the size of the fish or the weight of the fish? Do they receive the same dose or according to the ratio of body weight/dose?  Is there any difference between male and female fish for the results?

Line 237, missing full stop sign.

Figure 7: the magnification of the microscope need to be lableled.

Author Response

Comments 1: Line 104: why the 10% polyacrylamide gels instead of 4-12% gradient polyacrylamide gels? What is the quantity of protein sample was loaded into the gel?

Response: The 10 % polyacrylamide gel was chosen because of its efficiency in separating the proteins like immunoglobulin M. Protein load to the gel varied among the chromatographic fractions, but 8µl of protein sample containing 1-4 µg was applied to each lane.

Comments 2: Line 121: the quantity of the sample?

Response: 100µl of sample diluted appropriately in sodium bicarbonate buffer in case of the sensitivity assay of Anti-medaka IgM or BSA protein (1 mg/ml) in the case of immune response assay is added to 96 well microtiter plate. To address this issue, the sentence is corrected in the manuscript, line 119-120 “Sample was diluted in 50 mM sodium bicarbonate buffer (pH 9.6) and adsorbed in 96 well microtiter plate by incubating overnight at 4°C” was corrected to “Sample was diluted in 50mM sodium bicarbonate buffer (pH 9.6) and 100µl of this solution was added to a 96 well microtiter plate and incubated overnight at 4°C”

Comments 3: Line 140: in this section, what is the size of the fish or the weight of the fish? Do they receive the same dose or according to the ratio of body weight/dose?  Is there any difference between male and female fish for the results?

Response: Weight of the fish used in this study is provided in Table 1. Oral dosage of antigen was 10 µg per fish. Medaka gender was not taken into consideration in the study.

Comments 4: Line 237, missing full stop sign.

Response: Thank you for pointing out this problem. Full stop added in the manuscript.

Comments 5: Figure 7: the magnification of the microscope need to be lableled.

Response: Thank you for the valuable comment. We have added the magnification of the microscope in the relevant figure legend at Line 278-279.