The Antibody Two-Step Solution

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 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.  

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 http://f1000research.com/articles/2-153/v2 .  They argue that all publications using commercial antibodies should all 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.  This 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.

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

Why Recombinant Proteins Make Poor Antibody Validation Tools

Antibody validation is a topic that has garnered a great deal of attention lately in discussions of the problem with the lack of reproducibility in science.  One type of antibody validation that should be avoided uses purified recombinant proteins.  In a typical study of this type a purified recombinant protein is run on a western blot and then the labeling of the new antibody is examined.  Given that only a single protein is run on the blot, this “validation” study offers very little information about the specificity or sensitivity of the antibody.  The blot obviously has no information about whether the antibody recognizes other proteins since no other protein is present in the blot.  Moreover, absent any information about the relevance of the amount of recombinant protein used compared to the amount of endogenous protein in situ, the experiment does not even validate the ability of the antibody to bind to the protein of interest. 

Thus a western blot with recombinant protein does little to answer the two key questions about antibody quality: 1. ) Does the antibody possess the sensitivity to recognize the antigen in situ in a tissue of interest? and 2. ) Is the antibody binding specific for the antigen of interest in situ?  Nevertheless it is not unusual to see such data used to validate an antibody in product data sheets or even in refereed publications. So it is important to look carefully at any antibody validation blots to be sure that a cell lysate and not simply a purified protein is being analyzed.

In the example above I described a situation in which a recombinant protein based assay was used to give a false positive validation.  It is also not uncommon to see a recombinant protein based assay provide a false negative result i.e. to falsely invalidate an antibody.  Such experiments are most commonly seen with phosphospecific antibodies.  Such antibodies can be extremely valuable tools as they permit one to evaluate the phosphorylation state of a single phosphorylation site on a specific protein.  A critical question in the validation of such antibodies is whether they are indeed phosphospecific.  A mutant recombinant protein with the phosphorylation site of interested mutated to a non-phosphorylatable amino acid is run along side the recombinant protein in a western blot.  The antibody on interest is then tested for binding to this assay.  Binding to the non-phosphorylatable mutant in such an assay has been used by some as evidence that the antibody’s phosphospecificity has been invalidated.  Such data do not provide such evidence.  A phospho-specific antibody will always have at least a finite affinity for the non phospho-site.  Thus when, as is typical in such studies, micrograms or hundreds of nanograms of the mutant protein are run on the blot, some binding is highly likely.  In order to use such an assay for validation it is necessary to do a very detailed dose response with multiple concentrations of both the mutant non-phospho and the phosphoprotein.  Attention must also be paid to the concentration of the protein of interest in situ and also its level of phosphorylation at the site of interest.  Determining these values is always quite problematic.  Consequently the use of such a validation technique in not recommended particularly when other much more relevant validation assays are available.  The most common such assay is the western blot performed on lysates of the tissue of interest that had been incubated in the absence or presence of a phosphatase.  In such an assay the antibody is validated if it labels a single band in the control lysate and if the labeling is absent in the lysate that had been incubated in the presence of the phosphatase. 

One issue raised in the preceding paragraph was the fact that there is always a finite affinity of a phosphospecific antibody to the non-phosphoprotein.  Such binding of the phospho-antibody to the non-phosphosite can be quite problematic in negative affinity column selection of the phospho-antibody.  In our experience we have often found that very good phosphospecific antibodies may sometime fail to flow through a non-phospho column.  To avoid failures in such negative selection experiments it is very important to optimize the antibody to peptide ratio in using such a column.   This issue will be discussed in more detail in a subsequent blog.