Best Practices for Antibody Validation

Validating an antibody for research use involves far more than simply running a few cell lysates on a western blot and checking to see if a band of the correct size can be detected. Instead, robust antibody validation employs multiple testing strategies that, in combination, demonstrate specific binding to the protein of interest without any off-target effects. Although many antibody manufacturers perform extensive validation before releasing their products to market, it is critically important that researchers confirm antibody performance in their own lab. Here, four respected antibody suppliers share their validation expertise and provide advice for navigating the process of antibody selection.

Combining validation strategies builds confidence in antibody performance

“A well-validated antibody is one that, through the implementation of more than one approach, has been shown to recognize the target it is claimed to in a specific manner,” explains Katie Crosby, director of IHC at Cell Signaling Technology. “Effective strategies include binary validation, where the antibody is tested against samples that are known to express or not express the protein of interest, and ranged validation, involving the use of samples that exhibit high, moderate, and low levels of the target. Ideally, protein expression should be verified by an orthogonal approach such as in situ hybridization or mass spectrometry (MS), while testing data can be further strengthened by comparing the performance of two or more antibodies that recognize distinct, non-overlapping epitopes on the same target.”

Dr. Will Howat, director of antibody validation and characterization at Abcam, reports that the discovery and subsequent implementation of CRISPR/Cas9 into mainstream biology has provided a powerful means of determining true antibody specificity. “Knockout (KO) cells and cell lysates are valuable negative controls in the antibody generation pipeline that provide definitive confirmation of antibody specificity, particularly when performed in the application of interest,” he says. “However, one of the key steps in this process lies in confirming the validity of the KO cell line itself. This can be achieved by sequencing and may be followed by proteomic validation of the cell line using established antibody reagents. Where such antibodies have already been KO-validated, follow up studies provide even greater confidence that an antibody is specific for its target.”

“I would strongly encourage researchers to familiarize themselves with the five pillars of antibody validation,” notes Steve Orstad, product manager for R&D Systems brand antibodies at Bio-Techne. “These comprise genetic, orthogonal, and independent antibody validation strategies—as have already been mentioned—in addition to capture MS validation (where proteins captured by an antibody are subsequently identified by MS) and the expression of tagged proteins. Tagged proteins are used as standards in techniques such as immunocytochemistry (ICC), where the signal they produce is compared to endogenous target detection by the test antibody. In this situation, an overlap is considered indicative of antibody specificity.” Treatment-based approaches also enrich antibody validation data, for example by proving that a particular post-translational modification is detected.

Application-driven validation is essential

Just because an antibody performs in one application, there is no guarantee that it will perform in another. “In every application, the target protein presents itself in a different form,” reports Deepa Shankar, chief scientific officer at Proteintech. “For example, an antibody that detects a clear band on a western blot where the sample has been reduced and denatured may be unable to recognize the target protein under native western blotting conditions or in an application such as IHC. This difference in performance does not constitute a ‘bad’ antibody; rather, it highlights the importance of choosing antibodies that are suitable for your intended application.”

Image: IHC validation. Immunofluorescence analysis of human tonsil tissue stained with anti-CD3 antibody (green, 17617-1-AP) and anti-CD20 antibody (red, 60271-1-Ig). Image provided by Proteintech.

It is also worth keeping in mind that target-specific antibodies are but one component of an immunoassay. Other factors that should be considered include the sample-preparation method, the types of buffers used for blocking and washing, the incubation time and temperature, and the method of detection. In many cases, this information is included on antibody datasheets alongside the results generated where testing was performed under these conditions.

Validation begins with the antibody manufacturer

While antibody validation is largely viewed as being the responsibility of manufacturers, researchers are increasingly aware that they too have a major role to play. “Antibody manufacturers test antibodies in common applications and make every effort to match their testing to the needs of the research community,” says Howat. “Yet research performed globally will invariably differ, making it essential that further testing is carried out in the final lab setting to optimize conditions and provide application-specific validation.”

Crosby echoes this point, mentioning that the degree of additional testing the end user must perform will be influenced by the data supplied by the manufacturer. “An antibody provided with a thorough validation data package may need just a quick check in one’s lab, whereas an antibody with scant evidence of specificity likely requires a more comprehensive analysis,” she says. Shankar advises reviewing the antibody manufacturer’s information thoroughly and suggests that the more time researchers invest in looking for the right antibody, the less time they may need to spend on evaluating it in their chosen application.

Finally, Orstad comments that antibody manufacturers are keen to share their knowledge with researchers. “Because the COVID-19 pandemic has impacted researchers’ ability to be in the lab full-time, getting high-quality experimental results at the first attempt is even more critical,” he says. “As well as providing data demonstrating antibody specificity, many manufacturers offer a wealth of online resources, backed by technical support teams who can give expert advice.”

Tips for selecting the right antibody for your research

The following suggestions were pooled from all four contributors to this editorial:

• Look for antibodies that have been tested in your chosen application, ideally in the same species you will be using
• Review antibody datasheets for images that offer visual confirmation of testing and then critically evaluate the data—for example, check that the signal distribution and cellular localization in ICC/IHC aligns with expected results
• Try to use antibodies that have been validated using multiple approaches
• Where possible, choose antibodies validated in systems expressing the target protein at native/baseline levels
• Consider using KO-validated antibodies where available
• Where target homology with other family members is high, choose an antibody that has been proven to have no cross-reactivity
• Check antibody datasheets for details of experimental conditions, sample types, and controls used during testing; use these as a guideline for performing further titration or validation in your own experimental setting
• Never be afraid to ask antibody manufacturers for their advice
• Understand the vendor’s return and replacement policy—for example, find out what guarantee they offer that an antibody will work in a stated application or species
• Remember that the more time spent looking for the right antibody, the less time may be required to validate it in-house