Antibody Variability

In a recent article in Nature Methods, Vivien Marx discussed some of the key issues relating to the use of antibodies http://tinyurl.com/osygswn . 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. 

There are a number of reasons for the variability in a commercial antibody's performance.  The first is that 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 co-founder John Haycock made to tyrosine hydroxylase (TH).  This is our best selling antibody and we have been selling it for almost 25 years. 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 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. 

However, 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.

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 ever released. 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.

Now of course the next issue is whether or not the antibody with a given catalog number is actually specific for the target of interest.  This is a very interesting area and will be the topic for some of my next posts.

Antibodies That DO NOT WORK in Western Blots

What does it mean when an antibody company says an antibody “does not work” in western blots?

I started wondering about this question the other day after a colleague told me about a company that “validates” all of their antibodies by immunostaining in no less than 5 different tissues!  We examined the company’s website and in some cases western blots (WB) showing labeling of a single band were also presented.   But for the vast majority of products on the site only the apparently innocuous phrase “does not work in WB” was seen.  What does this mean?  I think for many antibody users (especially people who do not do WB and use various immunostaining protocols like IF or IHC) this failure to “work” in WB is often interpreted as meaning something akin to “don’t worry about the WB data, see if the antibody ‘works’ in immunostaining.”  However, I think any user of an antibody that “does not work in WB” should be very worried indeed.  This is because in the overwhelming majority of cases “does not work in WB” means the antibody labels many different proteins in a WB.  It is extremely rare to get an antibody that labels nothing in a WB. 

 Examples of antibodies that do and do not work in WB are shown in Fig. 1.  The lane at the right shows staining of a brain lysate with an antibody raised against tyrosine hydroxylase (THRAB) and it shows labeling of a single band of Mr 60,000 which is the approximate Mr of TH.  The lane at the left shows staining of an antibody that does not work in WB.  The antibody shown was raised against a protein Sap 102.  As can be seen in the image of the WB, the antibody does recognize a band at the appropriate Mr for Sap 102, but the antibody also recognizes many other bands as well.  This antibody ”did not work in WB” and it was thrown in the trash where it belonged.

So remember when you read the words “does not work in WB” you should translate that as “this antibody probably cross reacts with a number of different proteins.”