IgG Antibodies

Using the antigen as a blocking control to test antibody specificity can be problematic if an epitope is shared across multiple proteins.

Contributed by Mike Browning

Confirmation of antibody specificity is critical, especially in imaging assays such as IHC and IF.

How does one determine that the antibody specifically recognizes only the target of interest? There are a number of control procedures one can use to be sure that the signal generated in the antibody-based assay truly and quantitatively represents the presence of the target of interest. 

In western blots one can at least partially address this issue by determining that the relative molecular weight of the antibody signal matches that of the target. However in most other antibody based imaging assays (e.g. IHC and IF) no such information is available and thus determining specificity in such assays is even more critical.

Antibodies that only recognize a single epitope may seem "specific", but can cross react with other proteins that contain the epitope, causing a strong signal in IHC that would also be completely blocked by the epitope-containing antigen.

One of the most common controls for antibody specificity utilizes the antigen that was used to make the antibody as a blocking control. Unfortunately the value of this control is often greatly overestimated. For example, take a case where an antibody raised against a protein antigen recognizes only a single epitope in the protein. Assume for example that this antibody is non-specific and its epitope is also found in a number of other proteins. The antibody will thus recognize its epitope in all of those other proteins as well as in the target protein and thus in IHC it may give a very strong signal as it is detecting many proteins in the tissue. When one adds the immunizing antigen (which contains the epitope) to the antibody labeling assay, the antigen blocks the antibody labeling of all the proteins which contain the epitope. Thus it gives a complete block of all IHC signal. Normally that is interpreted as indicating that the antibody is specific. Clearly in this hypothetical case the blocking control failed because in fact the antibody was NOT specific.  

Non-specific antibodies can be more easily visualized in Western blot because of the molecular weight separation. 

peptide block in WesternThis effect can be seen in the Western blot at right. In  lane 1, an antibody raised against synaptotagmin labels three unknown protein bands in addition to the 60k band representing synaptotagmin. When the blocking control is used (lane 2) the labeling of the specific 60k band and all three non-specific bands is blocked.  So the blocking control eliminated all of the antibody signal but the antibody was clearly not specific for synaptotagmin. Thus anytime an antibody is non-specific and recognizes an epitope that is present in more than one target, the antigen blocking control is virtually useless. Since this type of cross reactivity or non-specificity is the one of the most troublesome types of antibody non-specificity, I would argue that antigen block is only one control to be used and that it is a relatively weak control for antibody specificity.

Two great controls for antibody specificity are tissue without the target antigen and tissue treated with phosphatase.

One of the best controls for antibody specificity is recombinant tissue that has been engineered to lack the target antigen. When using such tissue one should see no antibody signal in contrast to wild type tissue. Phosphatase-treated tissue is another one of the best controls is to use when testing phospho-specific antibodies. Provided that the phosphatase can dephosphorylate the target, the signal from a phosphospecific antibody should be eliminated from the phosphatase treated tissue with no change in the total amount of the target protein compared to untreated tissue.

Further Reading

Antibodies That DO NOT WORK in Western Blots

Are Monoclonal Antibodies Really More Specific?

Why Recombinant Proteins Make Poor Antibody Validation Tools

Antibody specificityPhosphosolutions