Antibody Validation 101

blog / Immunology October 30 2019

Many researchers start their hunt for new antibodies by scouring the literature to find out which antibodies others are using for the same target. While this is a reasonable starting point, simply choosing an antibody with reported target specificity and sensitivity is not enough, even if the published data looks very convincing! In practice, the suitability of an antibody for a given sample type, experimental conditions, and application depends on many more parameters than affinity for a target protein.

Ensuring that an antibody will yield reliable, accurate and reproducible results for every intended sample type and application is what antibody validation is all about.

Will Antibodies from Known Suppliers Always Be Validated?

Yes, and no. Antibody manufacturers use different protocols to validate their antibodies; some are very thorough in this process while others are vague. In either case, you will find validation details on the product datasheet. Standard validation approaches include western blotting, flow cytometry, ELISA, and immunohistochemistry. At least one of these should be carried out by the manufacturer and detailed in the datasheet. If you are in doubt, you can always reach out to your supplier to ask about other applications and sample types; it may be that they have other non-published data that could help you out.

Validation is time-consuming, because every parameter such as sample type, condition e.g., temperature, pH, denatured/native state, incubation period, and application must be assessed on a one-by-one basis. So don’t ever assume that an antibody that is validated in a few applications will work in another similar application.  Let’s have a look at the different ways in which antibodies can be validated, either by manufacturers or end users.

Good Ol’ Western Blot

The majority of manufacturers demonstrate that their antibodies ‘work’ by providing western blot results from whole cell lysates. But if this is the only validation data available, then you should proceed with caution.

A western blot may reveal a band at the anticipated molecular weight but it says nothing about the identity of the detected protein nor does it eliminate the possibility that the antibody has affinity for more than one protein in the same size range. Another drawback is that you may see additional bands at different sizes, but you’ll have no way of knowing with western blotting alone whether these represent non-target proteins or conformations of your target with post-translational modifications.

It’s not all doom and gloom though. While western blotting alone is not sufficient to validate a new antibody, it can be very useful when combined with other techniques as described below.

Knock It Out or Knock It Down!

Theoretically, gene knockout or deletion in your organism of interest should result in complete absence of the corresponding protein from subsequent samples. Where this is not possible, either because protocols don’t exist for the organism you are working with or because you’re working with an essential protein, gene knockdown by RNA interference (RNAi) is a good alternative. Using this approach, wild-type samples are run side-by-side with the mutant (i.e. the knockdown or knockout samples), usually in a western blot, and if the antibody binds the intended target, a signal will only be seen in the wild-type lane.

Manufacturers who perform this kind of validation will include the data in the datasheet. If this hasn’t been done for your chosen antibody and you want to do it yourself and you do see a band in your knockout/-down lane, then you should perform RT-PCR to ensure that the knockdown or knockout actually worked before you discard an antibody that might have potential!

Compare Independent Antibodies Against the Same Target

Comparing two independent antibodies raised against the same target protein is an easy way to assess antibody specificity, and this approach can easily be applied to many applications. Manufacturers who perform this type of validation usually compare their own antibody with that of an unidentified competitor.

To get the most out of your efforts if doing this yourself, you should include at least two antibodies that ideally recognise different epitopes on the same target protein, across several samples and sample types with varying expression levels of the target protein. This way, you gain information about specificity and sensitivity as well as potential clues about dilution factors to use in samples with low or high target abundance.

Overexpress or Perturb Your Target Protein

To confirm that an endogenous western blot signal actually comes from the target protein, manufacturers often perform overexpression experiments as a supplement to other validation approaches. Here, the manufacturer’s chosen organism/cell line(s) are transfected with a plasmid that encodes the target protein. Then, wild-type and transfected samples are run side-by-side in a western blot and the signals compared. If the antibody under investigation is specific, the transfected sample should yield a more intense signal than the wild-type.

If you are performing this type of validation yourself and have a deep understanding of how your protein is regulated, you may be able to perturb its expression levels more ‘naturally’ by altering environmental or nutritional conditions rather than transfecting with foreign DNA. Alternatively, you may expose your cells or samples to drug treatments or subcellular fractionation (e.g. if you expect your target protein to be in the nuclear fraction, in which case you should not see a signal in the cytosolic fraction). Some researchers also choose to compare results in cell lines with varying target expression levels (as confirmed at the mRNA level). In all cases, the signal intensity should go up or down according to your protein’s expression level.

If you do choose to overexpress with a genetic vector, you may want to titrate the amount of vector you use and check that the signal intensity goes up or down accordingly. Bear in mind that the intense signals generated by overexpressed proteins can mask off-target binding, so it is not worth attempting to validate an antibody in this way unless you see at least some endogenous signal in your sample type(s).

Immunoprecipitation or Mass spectrometry Analysis

Mass spectrometry (MS) is a powerful means to validate antibody specificity and performance by identifying the protein sequence(s) bound by an antibody. Here, protein lysates are typically incubated with the antibody under investigation (immunoprecipitation) and the proteins that remain bound to the antibody after washing are enzymatically separated from the antibody and identified by mass spectrometry. It’s worth noting that this approach may be complicated by indirect binding of protein(s) to an antibody through the interaction of the target with other proteins. Therefore, as with the other validation strategies, MS is best used in combination with some of the other approaches.

Standardised Antibody Validation

Many scientific journals and funding agencies now request information about how antibodies are validated by researchers, so you should always consider this before getting too deep into your research project. Generally speaking, the more ways a new antibody’s performance can be validated the better. You can save yourself some time and money by choosing to buy antibodies from manufacturers that use rigorous validation protocols, but in most cases you will still need to validate the antibody for your own sample type and experimental conditions.

Right now, there is no universal set of guidelines for antibody validation. However, a number of international working groups are attempting to change this, so who knows, maybe we will see some hard and fast rules one day soon!

Related Resources:

  1. Uhlen et al., A proposal for validation of antibodies. Nat Methods 13, 823-827 (2016).
  2. Bradbury, A. Plückthun, Reproducibility: Standardize antibodies used in research. Nature 518, 27-29 (2015).
  3. Information about the Antibody Validation Initiative (Global Biological Standards Institute Website).