Not all antibodies are valid for every experiment and condition, they must be validated for the specific application and species. Currently, there is no standard means of "antibody validation", and this can greatly impact experimental reproducibility and reliability. Journals and granting agencies have taken steps to address this gap. Many now have requirements to explicitly state how you will validate an antibody for a specific use. Unfortunately, there are no universally accepted criteria for antibody validation. So, it is up to each researcher to validate each and every antibody for the intended application.
Standard antibody validation methods include western blot, ELISA, flow cytometry, and IHC. Most researchers are comfortable with these tried-and-true methods. The validation process can be time-consuming because the researcher or the manufacturer must validate the antibody for each application. This difficulty is compounded because each the conditions of each assay are different. For instance, a western blot depends on the denaturing of proteins. So, a western blot-validated antibody may work fine in denaturing conditions but may fail to recognize antigens in their native conformation (i.e., ELISA).1 Likewise, an antibody validated for native protein affinity could fail to bind the same antigen following denaturation or fixation.
The above methods play necessary roles in validating antibodies. However, we cannot rely solely on these methods, because they do not represent the ever-expanding applications. The confidence level of the validated antibodies can be increased by including other validation techniques.
Old Techniques, New Uses
To address the short-comings of basic antibody validation, a group of scientists recently gathered to "propose a set of standard guidelines for validating antibodies".2 They suggest that in addition to basic antibody validation methods, researchers begin to lean on conceptual pillars. These include genetic strategies, independent antibody strategies, and tagged protein expression.2
Genetic Strategies: CRISPR-Cas9 and More
Genetic strategies consist of techniques like CRISPR-Cas9, RNAi, and siRNA knockdown, used in conjunction with a protein detection assay. These methods detect any non-specific binding by the antibody in question after knocking out or down the appropriate gene. If the antibody is specific, then no or reduced signal from the antibody should be detected in the knocked out or down cell lines, respectively.
Independent Antibody Approach
The use of independent antibodies is another method that could detect non-specific binding. The independent antibody approach employs two different (independent) antibodies that bind the same antigen but different epitopes. Therefore, the two antibodies should exhibit the same detection pattern and no off-target binding.2 This technique requires a multiple samples for testing to control for variable target protein expression, which could become expensive and time-consuming.2 Additionally, the validation technique relies on the availability of multiple antibodies that recognize different epitopes on the same target protein. GenScript offers epitope binning in MonoExpress™ mAb services which include “independent antibodies” for protein antigens.
Tagged Protein Expression
Analyzing the detection of tagged target proteins may also provide a way to measure the non-specific binding of antibodies. In this method, target proteins are modified with either an affinity tag (i.e., FLAG) or a fluorescent protein (i.e., GFP). Then, similar to the independent antibody approach, the detection pattern of the antibody being validated is compared to that of the tag-specific antibody. While straightforward, one issue with this method is ensuring that the tagged proteins are expressed at endogenous levels, because "overexpression could mask the detection of off-target binding events".2
Recombinant Antibodies as an Alternative to Monoclonal Antibodies?
A recent call to action regarding the validation of antibodies suggested that researchers use only recombinant antibodies, rather than polyclonal or even monoclonal antibodies. Monoclonal antibodies are produced using hybridoma cell lines. Unfortunately, hybridoma cell lines can die off, lose their antibody-encoding genes, or not grow when taken out of frozen storage.3 Moreover hybridoma-produced monoclonal antibodies could bind to more than one target. Therefore, determining the sequence of the antibody encoded by the hybridoma and producing the antibody recombinantly overcomes these problems. Also, because of the defined sequence, recombinant antibodies can provide another layer of validation compared to using the above techniques alone.3
These additional validation methods propose to "provide evidence that an antibody binds its target, and in most cases, they should also allow evaluation of potential cross-reactivity under the conditions tested".2 However, old and new strategies will likely need to be performed in combination to properly validate antibody.
Choosing the Appropriate Antibody Validation Method
With all of these issues surrounding antibodies, how do you choose the appropriate method?
First, you need to decide in which assay you will use the antibody. For instance, the CRISPR-Cas9 method does not involve modification of the target protein and validates that the antibody lacks cross-reactivity with other proteins. So, you can use this technique to validate antibodies for a wide variety of assays, including western blot, IHC, immunocytochemistry (ICC), flow cytometry, ELISA, immunoprecipitation (IP), chromatin immunoprecipitation (ChIP), and reverse-phase protein assays.2 However, because of the difficulties with expressing a modified protein, validating an antibody using tagged protein expression is suggested for only western blots, IHC, ICC, and flow cytometry.
Second, the researcher must be aware of the sample limitations of such validation methods. As an example, both CRISPR-Cas9/KO and tagged protein expression techniques cannot be used to validate antibodies in human tissue samples and body fluids, such as plasma and serum.2 That is because you cannot perform genetic manipulation in humans, unlike in cell lines.
Finally, regardless of the antibody and respective validation method used by the antibody provider, the researcher must perform at least one validation strategy in their particular application or sample context.2
Validating each antibody for your specific application and species is key to achieving reproducible and reliable results. The information will help guide your decision as to the appropriate methods to use in your laboratory. For further help with antibody validation or other applications, visit GenScript Antibody Technical Resources.
Adapted from content created by BitesizeBio on behalf of GenScript.
- Bordeaux, J., et al. Antibody validation. BioTechniques. 2010.
- Uhlen M, et al. A proposal for validation of antibodies. Nat Methods. 2016.
- Bradbury A., et al. Standardize antibodies used in research. Nature. 2015.