Competition assays for antibody specificity validation

Aliyah Weinstein

Confirming the specificity of an antibody you’re using for an experiment is an important step in ensuring that the results you observe are due to specific binding to your target protein. Competition assays using the immunizing peptide are a common way to test for specificity, ensuring that the antibody can bind to the antigen against which it was raised. However, this does not confirm that the peptide represents an available binding site in the whole protein.

In contrast, using the whole target protein to block with has some significant advantages over using a peptide. Blocking with whole protein can ensure that the antibody is able to bind the protein in its native state. While further validation studies will be necessary based on the particular application you’re planning, testing the specificity of your antibody against whole protein instead of against the immunizing peptide is one component to validating your antibody. However, blocking with the whole protein requires additional considerations over using the peptide, too.

Experimental considerations

Notably, it’s important to properly calculate the antibody-to-protein ratio in setting up this experiment. You must calculate this based on the ratio of the molecular weight of your protein versus that of an antibody (a standard 150 kDa is often used), not based on matching the concentrations of each. This is because the molarity of a protein solution will differ based on the molecular weight of a given protein.

You must also factor the bivalency of an antibody molecule into your calculations. For each molecule of antibody present in your solution, you need, theoretically, two molecules of protein to block with, due to the two Fab regions on each antibody molecule (more on this below). In considering both the molecular weight of your protein of interest and the bivalency of an immunoglobulin, your calculations will ensure that for every molecule of antibody present in your solution, you will have, at minimum, two molecules of protein for it to bind.

Performing the calculations

First, you will need to calculate the molarity of your final antibody solution, using a volume and dilution you’ve previously optimized for your particular testing situations (whether based on the manufacturer’s recommended conditions or previous tests you’ve performed, for example based on detected signal intensity).

Next, you will need to calculate for each experimental molar ratio of antibody to protein. While (as mentioned above) the theoretical blocking ratio is two molecules of protein to one molecule of antibody, experimentally, it is optimal to test multiple ratios to increase the likelihood of antibody and protein molecules binding each other in solution. Ideally, it’s recommended to test antibody to protein ratios of 1:2, 1:5, and 1:10, as well as 1:0 (i.e. no protein added, as a positive control for your antibody) and an isotype control (negative control). You should see optimal blocking at an antibody-to-protein ratio of 1:5 or 1:10. At a molar ratio of 1:2, you’re counting on 100% of molecules in the solution binding, which is statistically unlikely to occur. At high concentrations of target protein, you will, in fact, begin to see the blocking effect taper off and a return of your positive signal. However, this test only requires your signal to be blocked at one ratio—typically observed at 1:5.

Once the aforementioned solutions are prepared, apply them in your experiment during the step when you would normally add the primary antibody. Follow the rest of your protocol as established for signal detection. However, note that if your primary antibody is too concentrated, you may not see blocking. Instead of assuming that your antibody is binding nonspecifically, test higher dilutions of your primary antibody, then repeat a blocking analysis at the most dilute concentration at which you can detect a signal.

Assay-specific validation

Of course, further validation should be done within the context of the particular assay for which your antibody will be used. For example, you may validate that your antibody recognizes a protein of the correct size (Western blotting) or that it is co-localized with an appropriate marker (flow cytometry or immunostaining). However, ensuring the specificity of your antibody for its target protein using the most robust technique possible is an important step toward making sure that you will obtain reliable results.

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Aliyah W.

Aliyah is a PhD candidate at the University of Pittsburgh, where she studies cancer immunology. She is also an advocate for science communication. You can find her on Twitter @desabsurdites and on her blog at

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