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Opinion Article
Revised

The Antibody Two-Step Solution

[version 2; peer review: 3 approved]
PUBLISHED 22 Dec 2015
Author details Author details
OPEN PEER REVIEW
REVIEWER STATUS

This article is included in the Antibody Validations gateway.

Abstract

Problems with antibody quality have been described in numerous recent publications.  In the present commentary it is argued that these quality problems are due primarily to issues of antibody variability and antibody validation.  Further it is argued that the problem of antibody variability must be solved before validation can be useful.  A two-step solution to the antibody problem is thus proposed.

Keywords

Polyclonal, Monoclonal, Antibody, Variability, Validation, Pooled Serum

Revised Amendments from Version 1

I have added a reference to the RRID process as an additional way to identify antibodies.  I added a sentence explaining this reference.

See the author's detailed response to the review by C. Fernando Valenzuela

In the past year there have been a number of articles in Nature and other major journals that discuss the antibody problem in somewhat apocalyptic terms1. In my mind there are only two main issues in the antibody problem: antibody validation, and antibody variability. Both of these issues have straightforward solutions that do not require any massive influx of cash or massive restructuring of antibody production.

Antibody validation is the hardest nut to crack and causes the most confusion. There is no consensus on what constitutes suitable validation and this is complicated by the different methods for antibody use. However, antibody validation is a process like all science where knowledge increases as more and more work is done with the antibody. Many journals now insist that antibody validation data be provided and this is a key part of the solution. As long as the data are clear and the methods used adequately described, progress in validation will occur with time. However, none of this progress will matter unless we deal with the antibody variability problem. What difference does it make if an antibody is validated if it is not possible to obtain the same antibody for future work?

There are two main reasons for the variability in an antibody’s performance. The first is that once an antibody is found to have a high demand, many different antibody manufacturers will try to make their own version of the antibody so they can sell it. But all these new antibodies will differ in unknown and unpredictable ways from the original antibody. Thus validation done on the original antibody may or may not be true for the new antibodies. One way to deal with this problem was recently suggested by Andrew Chalmers and his colleagues2. They argue that all publications using commercial antibodies should report the name of the supplier and the catalog number of the antibody used. That way even if a supplier sells many varieties of the antibody a researcher will be able to order the same antibody that was used in the publication. Subsequently Bandrowski et al.3 proposed an even more detailed and efficient antibody identification protocol with their Research Resource Identifiers (RRIDs) which are based on accession numbers assigned by an authoritative database. These suggestions are being incorporated into the instructions to authors in more and more journals.

Even though this action would greatly improve the value of antibody validation, an additional source of antibody variability would remain, namely lot-to-lot variability. This variability occurs because even if one buys the same antibody with the same catalog or RRID number, one still often encounters large variability in different lots of the same antibody obtained from different bleeds of the same animal or bleeds from different animals. There is a very straightforward fix to this type of variability. The solution is to pool all the positively screened serum collected from the animals. Virtually all lot-to-lot variability can be eliminated for polyclonal antibodies if this procedure is used. The antibody manufacturer could simply label the antibody as “pooled serum” to denote this fact. If this procedure is followed, it will no longer be necessary to reinvent the antibody validation wheel each time an antibody is used. Thus science can build upon itself as it is supposed to do.

Some may argue that one should use monoclonal antibodies to eliminate variability. This is unnecessary and also unwise. It is unnecessary because for most antibodies a single rabbit can produce a stable 20–30 year supply of antibody. Only a small percentage of all antibodies sold ever sell more than can be produced by a single rabbit. It is unwise because monoclonals cost at least 3X what polyclonals cost and we are unlikely to see a time in the near future when cost will be irrelevant. Moreover, polyclonal antibodies have been shown to be superior to monoclonal antibodies in a number of different applications4.

Scientists and journals can fix the validation problem if antibody manufacturers will first fix the variability problem. This is called the Antibody Two-Step Solution.

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Version 2
VERSION 2 PUBLISHED 15 Sep 2015
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how to cite this article
Browning M. The Antibody Two-Step Solution [version 2; peer review: 3 approved]. F1000Research 2015, 4:810 (https://doi.org/10.12688/f1000research.7055.2)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Open Peer Review

Current Reviewer Status: ?
Key to Reviewer Statuses VIEW
ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 1
VERSION 1
PUBLISHED 15 Sep 2015
Views
25
Cite
Reviewer Report 06 Oct 2015
Angus C. Nairn, Department of Psychiatry, Yale School of Medicine, New Haven, CT, USA 
Approved
VIEWS 25
This short commentary article presents an opinion on the ongoing discussion of quality control of commercial antibodies. This is an important topic given the importance of these reagents to progress in a wide range of biomedicine, and of the large ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Nairn AC. Reviewer Report For: The Antibody Two-Step Solution [version 2; peer review: 3 approved]. F1000Research 2015, 4:810 (https://doi.org/10.5256/f1000research.7595.r10697)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
Views
26
Cite
Reviewer Report 29 Sep 2015
Andrew D. Chalmers, Department of Biology and Biochemistry, University of Bath, Bath, UK 
Approved
VIEWS 26
The opinion piece by Mike Browning makes a clear proposal to reduce the problems associated with using research antibodies. He proposes a two-step process, where step 1 is to reduce variability and the second step is to improve validation. The ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Chalmers AD. Reviewer Report For: The Antibody Two-Step Solution [version 2; peer review: 3 approved]. F1000Research 2015, 4:810 (https://doi.org/10.5256/f1000research.7595.r10334)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Reader Comment 19 Oct 2015
    Mike Browning, PhosphoSolutions LLC, USA
    19 Oct 2015
    Reader Comment
    Many thanks for your comments Dr. Chalmers. You raise the issue of lot numbers and suggest that these numbers should also be recorded and reported. As I indicated in my ... Continue reading
COMMENTS ON THIS REPORT
  • Reader Comment 19 Oct 2015
    Mike Browning, PhosphoSolutions LLC, USA
    19 Oct 2015
    Reader Comment
    Many thanks for your comments Dr. Chalmers. You raise the issue of lot numbers and suggest that these numbers should also be recorded and reported. As I indicated in my ... Continue reading
Views
29
Cite
Reviewer Report 25 Sep 2015
C. Fernando Valenzuela, Department of Neurosciences, School of Medicine, UNM Health Sciences Center, University of New Mexico, Albuquerque, NM, USA 
Approved
VIEWS 29
The paper of Browning proposes a two-step solution to solve problems related to antibody performance in biomedical research. The author proposes that antibody variability must be fixed first (for instance, by pooling positive serum from multiple animals). Once this issue ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Valenzuela CF. Reviewer Report For: The Antibody Two-Step Solution [version 2; peer review: 3 approved]. F1000Research 2015, 4:810 (https://doi.org/10.5256/f1000research.7595.r10333)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 13 Oct 2015
    Mike Browning, PhosphoSolutions LLC, USA
    13 Oct 2015
    Author Response
    Many thanks for your comments Dr. Valenzuela. I share your frustration with the issue of antibody validation. However, if variability is not dealt with first, validation has little value to ... Continue reading
  • Reader Comment 14 Oct 2015
    Fernando Valenzuela
    14 Oct 2015
    Reader Comment
    I have no further comments.  I recommend final approval of this commentary.
    Competing Interests: None
COMMENTS ON THIS REPORT
  • Author Response 13 Oct 2015
    Mike Browning, PhosphoSolutions LLC, USA
    13 Oct 2015
    Author Response
    Many thanks for your comments Dr. Valenzuela. I share your frustration with the issue of antibody validation. However, if variability is not dealt with first, validation has little value to ... Continue reading
  • Reader Comment 14 Oct 2015
    Fernando Valenzuela
    14 Oct 2015
    Reader Comment
    I have no further comments.  I recommend final approval of this commentary.
    Competing Interests: None

Comments on this article Comments (0)

Version 2
VERSION 2 PUBLISHED 15 Sep 2015
Comment
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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