If you have suffered through western blot troubleshooting, you understand how vital your antibody supply is. The most common problems in antibody-dependent experiments are when the antibody binds off-target proteins or when the conditions in the cell or lysate under study differ from those the antibody was originally validated in. Tremendous amounts of work are needed when using a new antibody to determine the conditions for its use for each individual experiment. The last thing you want to do is waste those hours and days with an antibody that isn’t reproducibly specific, selective, or sensitive enough. When choosing an antibody, ask the manufacturer what validation pillars they use to assess the quality of their antibodies before you get them.

Pillars for antibody validation

In 2016, an international group of scientists, who were frustrated with the lack of consistency in antibody production, met and formed the ad hoc International Working Group for Antibody Validation (IWGAV). In their 2016¹ and 2018² publications, they established a list of validation approaches. At least one of these techniques should be used to validate antibodies before experimental optimization and use. 

Independent antibody studies

One common method to validate antibodies is to use more than one. A good manufacturer will create two or more independent antibodies to the same protein, but different epitopes. They can compare the results of both of these antibodies to ensure the data are similar.

Orthogonal validation

Comparing antibody-independent data with the results from antibody-dependent studies done during manufacture is another common validation approach. This approach confirms that the expression patterns seen with antibodies match up with what is expected from other techniques, such as proteomics or transcriptomics. 

Other approaches

IWGAV suggested three other approaches to antibody validation: genetic knockdowns to demonstrate the antibody recognizes the target protein in the presence of its intact gene compared to the knockdown; recombinant expression to show the absence of signal in a wild-type line compared to the presence of signal when using an expression vector; or immunocapture followed by mass spectrometry to confirm the antibody recognized the right protein. 

Trust your source

Ask your current antibody manufacturer how they validate their antibodies. Do they do the minimum or go the extra mile? Years before these pillars were published, Bethyl has validated their antibodies using a combination of orthogonal methods and independent antibodies. This culminated in the creation of the reciprocal immunoprecipitation method (see the figure). For proteins with difficult expression levels or patterns, they use protein overexpression or knockdown validations as well. Bethyl also uses unique biological characteristics of the target protein, such as differential expression or other alterations to validate antibody to target recognition. 

Validation methods are not universal for every type of antibody application. Optimization done with one method may not be applicable to another method. Bethyl uses extensive validation techniques such as complementary assays to compare results from more than one immunoassay, such as western blotting to immunocytochemistry. These processes validate the antibody, but they also help scientists by giving them a better starting point for their own optimization.

1. Uhlen, M., et al. A proposal for validation of antibodies. Nat. Methods 13, 823-827 (2016).
2. Edfors, F., et al. Enhanced validation of antibodies for research applications. Nat. Commun 9, 4130 (2018).