Identification of a New B-Cell Epitope on the Capsid Protein of Avian Leukosis Virus and Its Application

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Wang et al. found that the monoclonal antibody 1F8, generated from mice immunized with ALV-J, recognized amino acids 218-227 of p27 capsid protein. This epitope is found to be conserved in ALV-A, B, J, and K. Although the newly discovered B-cell epitope of capsid protein is not suitable for vaccines, it is effective for diagnosis based on various clinical materials and is expected to be used in eradication strategies by detecting ALV-infected chickens. The manuscript was carefully written and includes valuable information. 

However, the validity of the sandwich ELISA constructed by the authors needs to be explained more emphatically. For this reason, I think it is necessary to indicate in the manuscript how the antigen levels of the various avian pathogens used to show that the sandwich ELISA is specific for ALV were standardized. In addition, it is necessary to explain whether the cloaca swab that tested positive in the sandwich ELISA is a true positive specimen.

 

The others are minor points; 

 

Materials and Methods

Line 81. Please state the concentration of IPTG.

Line 87. Is Sigma (USA) the manufacturer of “P27”, or is it the manufacturer of “Freund's adjuvant”?

Line 88. Balb/C mice -> BALB/c mice. In line 100, you described as BALB/c mice.

L137. rosetta (DE3) -> Rosetta (DE3)

Line 92. What exactly is shock immunization?

Line 142. What method was used for the peptide ELISA; if it is the same as the indirect ELISA described in 2.6, why is it measured at 450 nm?

Lines 158-159. Please provide details on various viruses or samples (avian viruses and bacteria). How each sample was prepared and how antigen levels were standardized for sandwich ELISA should be indicated. M. synoviae, M. gallisepticum, Escherichia coli, Salmonella, Pasteulella should be written in italics.

Result

 

-The description in Figure 1(E-F) is not accurate. First, the antigen applied to the gel in Figure 1E is not a positive serum or negative serum. The description in the figure should be changed. Next, we cannot be certain that Figure 1F is the western blotting image stained with the anti-His tag antibody. Legend of Figure 1 should be revised. For example, Figure 1E and F should be explained separately.

 

-The letters in Figure 6A are too small to read. Reading the explanation in the text about Figure 6A, it is not at all clear what it means, so the image must be improved.

-I do not understand the significance of the sentence “The sandwich ELISA has a higher positive detection rate of ALV in cloacal swabs (line 295). This is because the author did not provide information if the cloaca swab that tested positive in the sandwich ELISA is a true positive specimen.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors generated hybridomas secreting MAbs against the capsid protein (P27) of ALV. They characterized one specific MAb (1F8). They determined its binding affinity and confirmed that it could be used to detect multiple ALV strains (A/B/J/K). They also mapped the B-cell epitope present on P27 and developed a sandwich ELISA using 1F8 to detect ALV antigens.

My critiques and suggestions for improvement follow:

1) Line 182: It is not clear what the authors mean by "bitter ammonium sulfate law".

2) Figure 1, panel B: It is not clear which bands the authors used to confirm the pET-28a-P27 plasmid. The authors should indicate the bands and the RE they did to help the reader understand.

3) Line 192: The authors mention a previous study but provide no citation. Please provide a citation for the previous study.

4) Figure 3, panel A: The IFA images are unclear and difficult to evaluate. At a minimum, the authors should indicate a scale for this image. Ideally, the authors should magnify sections of the IFA to allow the readers to better see the staining.

5) Figure 3, panel B: What is the band running at 55 kDa?

6) Figure 4, panel B: the authors should indicate the expected size of each of the peptide constructs on the SDS-PAGE gel. 

7) Figure 4, panel B, Western blots: The anti-GST and 1F8-MAb bands are very weak, while the background is very high. Thus, it is difficult to evaluate these Western blots. I urge the authors to include better western blots, especially for the Anti-GST. It is also suspicious that the different peptide constructs all have the same migration pattern when detected by anti-GST. Shoud not the different constructs have different sizes?

Comments on the Quality of English Language

While the paper was easy to understand, the manuscript would benefit from a good English language editor to eliminate numerous grammatical errors and improve vocabulary.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This is a standard B-cell epitope paper by Wang et al., belonging to a group in China, where the poultry industry plays a big role in public nutrition and economy. It is scientifically complete, simple, but unremarkable. Developing a monoclonal against an epitope is routine work these days. Most laboratories do not even bother to publish them. However, it is interesting nonetheless.

I have a few minor comments / suggestions:

1) Like all scientists, the authors are understandably proud of their work; however, in the bigger scheme of things, there is really nothing “novel” in this paper. So, replace all “novel” with “new”. Anything that is “new” is not necessarily “novel”.

2) Figure 3 legend: for the general readers, it will be useful to add that FITC is green, and DAPI is blue, stains the nuclei. The easiest place is: (A) IFA (FITC = Green); DAPI (nuclear stain = Blue).

3) It is indeed clear that the predicted 3D structure in Fig. 6 shows the surface location of the authors’ immunologically validated 218-227 decapeptide. This raises a query. As the authors correctly noted (Fig. 7), the whole area from 181-227 “appears to encompass all the identified epitopes, suggesting its potential as a prominent B-cell epitope domain, which is also reasonable. But is this whole sequence also surface-exposed? Discussing this will be useful. The authors could, for example, put a different color, other than Red (Blue or Green) on the amino acids 181-218.

 English problem:

English needs to be improved in may places, particularly in the Discussion. A few examples (but the whole paper needs to be carefully read and such corrections made):

Line 351: and can more easily to be chimeric

Line 353: was remarkable conservation
Line 356: Delete “prepared” (since there is no such thing as “unprepared” antibody!). Maybe the authors wanted to write “purified”?

Comments on the Quality of English Language

See the last part of the comments for authors.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf