Epitope-Specific Anti-SerpinB3 Antibodies for SerpinB3 Recognition and Biological Activity Inhibition

Round 1

Reviewer 1 Report

This work describes the design and synthesis of selected peptides to raise anti-serpin B3 antibodies, as well as the characterization of the different antibodies produced against the selected peptides. The rationale behind the work is well described and the results presented deserve to be published, since they should be interesting for other researchers in the field, but some aspects of the work need to be improved. My suggestions are as follows:

 

- The manuscript needs careful reading to correct some English errors and sentences that are difficult to understand, in particular (but not exhaustive):

Abstract:

‘This antibody was able to recognize SerpinB3 at nuclear level, while with anti-P#3 antibody SerpinB3 only cytoplasmic staining was detected by both immunofluorescence and immunohistochemistry.’

‘… reduced of 12%’ (should be reduced by 12%)

Introduction:

‘The first is known that inhibits papain-like cysteine 40 proteases’ should be ’The first is known to inhibit…’

Chymo-trypsin should be chymotrypsin

Internal organ should be internal organs

Western blot should be western blot (no capital w); Dot-Blot should be dot blot; in general, capitals should be used only when needed.

 

- There is no explanation about why the authors decided to mix four of the peptides in two pairs for immunization and later immunopurified separate oligoclonal antibodies using the individual peptides; the reason for not performing separate immunizations should be discussed.

 

- Statistical analysis: there is no description in the methods or figure legends of the statistical test applied (Fig. 6B); figure 6A does not report any statistical analysis (if non-significant, it should be reported in the legend); figure 6A and B show essentially no standard deviation or error, the bars are almost invisible: how is it possible that all the repeats were identical?

 

- Figure 2A: the histogram doesn’t provide a complete information about the performance of the purified antibodies against the different antigens; a graph reporting curves with different concentrations of the antibodies in increasing dilutions would be much more informative.

 

- Figure 2B: this ELISA is differently explained in the methods, main text and figure legend, and is named in different ways (indirect and inverse) in the main text and in the figure. The protocol should be correctly described in the Methods section, reminded if needed in the figure consistently with the description given in the Methods section, and the correct term for this type of ELISA should be used (indirect).

 

- Figure 3 is cited in the text before figure 2B, this should be avoided: the figures should be cited in order, this requires changing either the figures or the text accordingly.

 

- Figure 4: insets at higher magnification are needed to evaluate the staining described in the main text (cytosolic, nuclear); a negative control image is also needed.

 

- Figure 5: the final magnification should be reported for all panels, ‘original magnification 63x’ is not correct.

 

- In the discussion, line 370, the authors state that ‘the anti-P#5 antibody was specifically reacting only against SB3’ which is not correct since the anti-P#5 antibody reacts with human SB4 in ELISA (figure 2A). This should be correctly discussed.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

I would like to congratulate the authors of the article, follow some suggestions to the manuscript.

- In line 276 - choose "r" or "R" to describe the recombinant serpin.

- In figure 3 and possible in other parts of the paper, use dot instead of comma, in the numerals.

- line 287 add one space after the 0.5

- figure 4 - add scale bar into the images. Indicate in the images the antibody marked structures.

- In statistical analysis describe the statistical tests used to evaluated the data.

- line 333 - "Figure 6 panel B" instead of Figure 5.

- It was not clear about the result of figure 6A if the statistical analysis indicated any differences among the groups.

-  in my opinion, in IHC and IF it was necessary to add a control demonstrating that the molecules recognised by anti-SB3#5 in the cell nucleus are really SerpinB3. One suggestion to do this is to use the anti-SB3/4 antibody, with another fluorescent marcking.

 

 

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have addressed all the issues raised and the manuscript is now suitable for publication.

Reviewer 2 Report

The article was modified according suggestions.