Antibody variability can cause frustration and irreproducible results. Lot-to-lot inconsistency in polyclonals can be addressed directly through serum pooling.
Contributed by Mike Browning
There are a number of reasons for the variability in a commercial antibody’s performance.
In a recent article in Nature Methods, Vivien Marx discussed some of the key issues relating to the use of antibodies. Variability in the quality of the antibodies available commercially was one of the principal problems encountered by scientists in her article. This problem is closely related to the larger issue addressed in numerous editorials in Nature and other journals regarding the irreproducibility of research.
Once an antibody is found to have a high demand, then many different antibody manufacturers will try to make the antibody so they can sell it.
As an example take the antibody our colleague and PhosphoSolutions' co-founder John Haycock made to tyrosine hydroxylase (TH). This is our best selling antibody and we have been selling it for decades. When John first started selling this antibody it was virtually the only TH antibody on the market. However today if one examines any large antibody company’s offerings one can find hundreds of sources of this TH antibody. So this is one huge contributor to antibody variability; namely that many different groups have made many different TH antibodies. So simply saying you used a TH antibody can mean you used any one of a huge variety of different 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 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.
Antibody signal strength can vary across different bleeds of the same animal.
As Ms. Marx points out in her article, 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. She quotes some antibody sellers who argue that this variability is unavoidable and simply the nature of the beast given that antibody quality can often vary across different bleeds from the same animal(s). An example of how the antibody signal strength and specificity can vary across bleeds from the same rabbit is shown in Figure 1.
Figure 1. Western blot of serum from bleeds 1-10 collected from a single rabbit. The level of immunoreactivity for the 210 kD band (see arrowhead) varies dramatically across the various bleeds. Moreover, the level of cross reactivity with bands of ~100 and ~50 kD also varies across bleeds albeit in a very different pattern than that for the 210 kD protein.
Pooling the serum collected from animals before it is purified can eliminate the variability of polyclonal antibodies.
We would argue that there is a very straightforward fix to this problem of variability. All it requires is some forethought and planning before an antibody is ever released. The solution is to pool all the serum collected from the animals before the first antibody is brought to market. Then all serum sold subsequently for that antibody catalog number will by definition be identical as it all comes from the same pool. Obviously in the serum shown in the figure, the antibody will have to be affinity purified. In such a case, all purifications will be performed identically with identical aliquots from the same pooled starting material. Thus virtually all lot to lot variability can be eliminated for polyclonal antibodies if this procedure is used. It should also go without saying that once the pool of antibody has been used up then that catalog number must be discontinued and not simply continued with antibodies from another animal.
The next issue is whether or not the antibody with a given catalog number is actually specific for the target of interest. See links below!
Further Reading
Antibodies that DO NOT WORK in Western Blots
Are Monoclonal Antibodies Really More Specific?
1% SDS is the Lysis Buffer of Choice for Most Western Blots