Antibody specificity:  The use of a blocking control has only limited value

Antibody specificity is one of the key issues in determining whether you have an antibody that works.  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 criticalOne 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.  
blocking peptide copyThis effect can be seen in the Figure at right.  In this western blot as shown 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.
            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.
Antibody Specificity:  The Use of a Blocking Control has only Limited Value
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Antibody Specificity: The Use of a Blocking Control has only Limited Value
Antibody specificity is often tested with the antigen used to make the antibody as a blocking control. However, if the antibody recognizes an epitope present in more than one protein, the antigen will also block any of these proteins that are present.
Mike Browning
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2 thoughts on “Antibody Specificity: The Use of a Blocking Control has only Limited Value

  1. Totally agreed. Blocking could be useful though when an antibody was raised against a full length protein and the full length protein (preferrably from a different purification from what was used for immunnization)is used for the blocking. Hopefully the strongest antibodies in the mix will be blocked by the protein while low affinity other proteins may not. Having said this, a much better control would be to check non-specific signals derived from the secondary antibody. This is often overlooked. Simply comparing the results with an identical experiment run in parallel without primary antibody will determine where any non-specific signal comes from. This is true for all types of antibodies.

  2. Hi Jan,

    Thanks so much for your comment. I totally agree as the secondary antibody control is so often omitted these days and as you say it is a much better way to examine the non-specific signals. Yours is the first comment to my new blog. So thank you again for your interest. I also liked reading your comments on the linkedin antibody group.


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