IgG antibody validation

Scientists and journals can fix the antibody validation problem if antibody suppliers will fix the antibody variability problem. We call this the Antibody Two-Step Solution. 

Contributed by Mike Browning

Two Antibody Problems: Validation and Variability

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 terms. 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 being addressed by both researchers and journals.

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. As long as the data are clear and the methods used adequately described, progress in validation will occur with time. Nature’s insistence in its instructions to authors that antibody validation data be provided is exactly the right path to be taken. However, none of this progress will matter unless we deal with the antibody variability problem.

Antibody variability can be addressed in two ways. The first is to include manufacturer and catalog number information in the Materials and Methods section.

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 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 not be true for the new antibodies. One way to deal with this problem was recently suggested by Andrew Chalmers and his colleagues in this article. 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. This suggestion is 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. 

Antibody variability must be addressed by antibody manufacturers.

Manufacturer-based variability occurs because even if one buys the same antibody with the same catalog number, one still often encounters large variability in different lots of the same antibody obtained from different bleeds of the same animal. There is a very straightforward fix to this type of variability. The solution is to pool all the serum collected from the animals. Virtually all lot–to-lot variability can be eliminated for polyclonal antibodies if this procedure is used. 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 20-30 year supply of antibody. Only 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. Only antibodies with a known, large market are likely to justify the monoclonal cost and maintenance expense.

The antibody "crisis" can be addressed if researchers and manufacturers work together and take responsibility. That is why we call it the Antibody Two-Step Solution.

Further Reading:

SDS-PAGE Demystified – The Science Behind All Those Bubbles!

Antibodies in Western and IHC

Antibody Variability

Are Monoclonal Antibodies Really More Specific?

1% SDS is the Lysis Buffer of Choice for Most Western Blots

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