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This paper uses fluorescently labelled DNA attached to streptavidin-coated magnetic beads to detect the presence of a biotinylated antibody on a surface. There's really no reason to use DNA, any fluorescently labelled biotinylated molecule would do fine. Also, the antigen is non-specifically adsorbed to the surface; a second antibody would result in a true ELISA antibody sandwich assay. Only one antigen is used; the specificity of the antigen-Ab binding is not confirmed. 这是什么意思啊？ |
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5楼2010-06-28 16:43:08
nandi2212
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2楼2010-06-28 16:24:14
xuezhaoteng
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Dear xx: The above referenced manuscript has been reviewed by three experts in the field. Reviewer #2 recommends major revision for prior to acceptance, while Reviewers #1 and #3 feel the manuscript is premature at the present stage. I think Reviewer #1 misunderstands the concept of the quantitative assay. I believe the quantitative measurements are made by the number of bright spots, not by brightness of each spot. However, Reviewer #1 does not highly appreciate the study. In light of these comments, we are unable to consider the manuscript further for publication in its present form. We would be willing, however, to consider a substantially revised manuscript that addresses these concerns. Please include a detailed cover letter that addresses each of the comments. Please pay special attention to some of the Reviewer’s comments as described below. Reviewer #3 raises very serious concerns concerning the concept of the study. The reviews are enclosed with this letter. (1) It is very essential to answer Reviewer #3’s concerns in a reasonable manner. (2)It is preferable to add in the text more comprehensive explanation concerning the quantitative methods and the control of biotin-DNA conjugate’s binding for evasion of similar misunderstanding by readers. Additionally, please make the following changes: Do not number the categories within the manuscript. This is not in the style of the journal. ***Note: Please return a document that contains both the text and graphics.*** To revise your manuscript, log into ACS Paragon Plus at http://paragonplus.acs.org/login and select "My Authoring Activity". There you will find your manuscript title listed under "Revisions Requested by Editorial Office." Your original files are available to you when you upload your revised manuscript. If you are replacing files, please remove the old version of the file from the manuscript before uploading the new file. When submitting your revised manuscript through ACS Paragon Plus, you will be able to respond to the comments made by the reviewer(s) in the text box provided or by attaching a file containing your detailed responses to all of the points raised by the reviewers. If your revised manuscript is received more than 90 days from the date of this letter, it may be considered as a new submission and and sent out for additional review. Thank you for your interest in Bioconjugate Chemistry and manuscript submission. Sincerely, Masayuki Yokoyama Associate Editor Bioconjugate Chemistry Email: Yokoyama-office@bioconj.acs.org ------------------------------------ Please click the link below to view the Bioconjugate Chemistry Author Guidelines: http://pubs.acs.org/page/bcches/submission/index.html Reviewer(s)' Comments to Author: Reviewer: 1 Recommendation: It appears that publication in any form would be premature at this time. Comments: This manuscript describes the development of quantitative detection of single molecules using enhancement of Dye-labeled nanoparticles. The idea is interesting, but this detection may not be quantitative. The number of biotin-DNA bound to avidin-beads cannot be normally controlled. In DNA hybridization, all complementary single-stranded oligonucleotides cannot be hybridized. Thus, this fluorescence immunoassay does not seem to be a general procedure for quantitative detection. Thus, it is not recommended for publication. Please rate the overall importance of this paper to the field of bioconjugate chemistry. (10 - High importance/1 - Low importance): 3 Reviewer: 2 Recommendation: Publish after major revisions noted. Comments: This paper describes a new methodology of signal amplification in single-molecule observations for highly sensitive immunoassays. The method is based on the in-situ preparation of antigen (IgG) / biotinylated antibody / streptavidin-coated magnetic nanobead / biotinylated double-stranded DNA / SYBR Green I conjugates on an epoxy-functionalized glass substrate. These conjugates are subject to epifluorescence microscopic observation with an electron multiplying CCD, affording the linear relationship between the number of magnetic beads and the concentration of antigen. Major and minor comments are as follows. Major points: 1. Compare the sensitivity, photostability, cost-effectiveness, etc of the present method with those of the authors’ previous methods (ref 23, 27 and 30) in the Results & Discussion part. 2. The experimental evidence for a 1:1 binding of a biotinylated antibody to a streptavidin-coated magnetic bead is not sufficient. 3. The origin of the quenching at ratio over 0.25 in Figure 4 is unclear. I cannot understand the reason why non-intercalative binding of SYBR Green I to dsDNA induces the significant decrease in fluorescence intensity. 4. In the legend of Figure 6, “(A1) 50, (B1) 30, (C1) 10, (D1) 8, (E1) 5, (F1) 3” should be “(A1) 3, (B1) 5, (C1) 8, (D1) 10, (E1) 30, (F1) 50”. In addition, the meaning of red circles in (F1) should be given in this legend. Minor points: 1. On page 8, line 3, describe the concentration of BT-Ab. 2. On page 8, line 14, add the information on the procedure for magnetic separation. The explanation on page 11, lines 6-9 should be moved to the Experimental Section. 3. On page 8, line 17, describe the concentration of BT-dsDNA. 4. On page 10, line 1, describe the calculated distance between proteins. 5. On page 10, line 3, impossible -> possible 6. On page 10, line 11, the distance between two adjacent MNBs -> the diameter of the MNB (?) 7. On page 12, line 2, 10^15 / M -> 10^15 / M^-1 8. On page 12, line 5, describe the value offered by the manufacture’s instructions. 9. On page 14, line 1 from the bottom, SYBR Green I/BT-dsDNA/SA-MNB -> SA-MNB/BT-dsDNA/SYBR Green I 10. … are shown in Figure 6, -> … are shown in Figure 6. (page 15, line 7) 11. … a 6 by 6 pixel area, While … -> … a 6 by 6 pixel area. While … (page 15, line 15) Please rate the overall importance of this paper to the field of bioconjugate chemistry. (10 - High importance/1 - Low importance): 6 Reviewer: 3 Recommendation: It appears that publication in any form would be premature at this time. Comments: This paper uses fluorescently labelled DNA attached to streptavidin-coated magnetic beads to detect the presence of a biotinylated antibody on a surface. There's really no reason to use DNA, any fluorescently labelled biotinylated molecule would do fine. Also, the antigen is non-specifically adsorbed to the surface; a second antibody would result in a true ELISA antibody sandwich assay. Only one antigen is used; the specificity of the antigen-Ab binding is not confirmed. Recommendation: reject and have the authors redo the work using a second surface antibody. Please rate the overall importance of this paper to the field of bioconjugate chemistry. (10 - High importance/1 - Low importance): 2 ----------------------------------------------------------------------- FOR ASSISTANCE WITH YOUR MANUSCRIPT SUBMISSION PLEASE CONTACT: ACS Publications Customer Services & Information (CSI) Email: support@services.acs.org Phone: 202-872-4357 Toll Free Phone: 800-227-9919 (USA/Canada only) 这是全部的审稿意见，大家帮忙看一下。 |
3楼2010-06-28 16:29:12
xuezhaoteng
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4楼2010-06-28 16:32:24
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