Antibody Validation Strategies

Within the past decade, countless publications have highlighted issues with antibody reproducibility.^1-5 As a result, researchers are more likely to question antibody quality and validation data. Here, we look at the effects of this changing mindset and speak with antibody manufacturers about some of the latest validation strategies being employed.

Antibody irreproducibility is driving change

“Every scientist I know, including myself, understands the pain of wasting a week on an experiment that failed simply because an antibody didn’t work as advertised,” says Benjamin Doranz, Ph.D., President and CEO at Cell Surface Bio (an Integral Molecular company). “However, researchers are now more aware of the issues with antibody reproducibility and this is driving change across the board. Not only are scientists becoming more careful when choosing commercial antibodies, but manufacturers are using more comprehensive validation strategies to evaluate their products.”

Unfortunately, inadequate validation is a leading cause of antibody irreproducibility. “Problems like cross-reactivity, batch inconsistencies, and use in inappropriate applications all contribute to irreproducible data, which can lead to paper retractions and flawed science,” notes Dr. Christian Frisch, R&D Manager for Custom Antibodies at Bio-Rad. “This has caused researchers to question the traditional antibody generation and validation methods, which marks a shift toward more rigorous validation processes and greater transparency in reporting. These changes are driving the scientific community toward better practices and more reliable, reproducible research.”

Proof of validation is essential

When reviewing an antibody’s validation data, knowing what to look for is key. While a solitary western blot showing detection of a recombinant protein may have sufficed before the turn of the century, modern-day researchers need more thorough proof that an antibody will perform as expected.

“Validation data should accurately define sensitivity, reproducibility, target specificity, and application specificity,” says Chandra Mohan, Ph.D., Technical Content Writer at MilliporeSigma (the life science business of Merck KGaA, Darmstadt, Germany in the U.S. and Canada). “It is crucial to note that not all antibodies will work with every application or react with each species—you should always confirm and validate an antibody for your own specific model system.”

Other types of validation to look for include functional testing and confirmation of antibody purity. “Functional assays like binding assays offer critical insights into binding activity, kinetics, and affinity, which are vital for accurate antibody characterization,” explains Frisch. “Verifying the purification level of antibody reagents using techniques like SDS-PAGE and chromatography is necessary to ensure high quality and avoid contamination.”

Sophisticated validation strategies

To ensure reliable antibody performance, manufacturers are using increasingly sophisticated validation strategies. “At MilliporeSigma, we validate our antibodies using multiple techniques,” says Mohan. “For example, we evaluate our ZooMAb^® recombinant monoclonal antibodies in a minimum of three different applications to ensure that all of those assays produce complementary results. For some antibodies, we may include genetic strategies, independent verification with multiple antibodies, and the use of RNA-seq to validate specificity. We also collaborate with academic researchers to independently evaluate our antibodies before we make them available for sale.”

Cell Surface Bio’s approach includes testing every antibody in its catalog on Integral Molecular’s Membrane Proteome Array™ (MPA), which covers all 6,000 native membrane proteins in the human genome. Binding is measured via flow cytometry in unfixed cells, ensuring native protein conformation. “About one-third of all monoclonal antibodies demonstrate polyspecificity and bind to unintended targets,” reports Doranz.⁶ “Often, those off-targets are not other family members, which are usually tested during discovery, but unrelated proteins that would be impossible to predict. By testing our antibodies against the entire human membrane proteome, we ensure an unprecedented level of specificity.”

Testing of the GLUT4 monoclonal antibody (CSB0138) on the MPA, which contains 6,000 different native membrane proteins, demonstrates high specificity. Image courtesy of Cell Surface Bio.

At Bio-Rad, DNA sequencing is used to validate antibody clones produced using the HuCAL^® recombinant monoclonal antibody library. “Our process generates fully human anti-biotherapeutic antibodies in Fab and full-length Ig formats for bioanalytical assays, using in vitro guided selection to ensure high specificity,” says Frisch. “Not only does knowing the antibody sequence allow for verifying the identity of the clone, but it also ensures its long-term availability.”

The critical role of antibody manufacturers

The question of who is responsible for validation has been debated in numerous editorials, committee initiatives, and panel discussions. However, everyone interviewed here agreed that the onus was mainly on manufacturers.

“Quality assurance and confidence in commercially available antibodies is something researchers naturally expect,” says Mohan. “Hence, it is the primary responsibility of commercial suppliers to validate and provide reliable data to support the performance of their antibodies. Having said that, the final decision regarding which antibody to use lies in the hands of researchers. We try to provide the best validation data and species reactivity information on our website and encourage researchers to contact our technical service scientists to help them make the right choice.”

Frisch adds that manufacturers have a central role to play in addressing the reproducibility crisis in scientific research. “Ensuring product quality is essential to scientific integrity,” he says. “Therefore, I believe that the weight of improving experimental reproducibility lies with the manufacturers, as they should be more rigorous when validating their products. Ultimately, manufacturers must lead in providing reliable, well-validated antibodies to support reproducible and trustworthy scientific research.”

Lastly, Doranz comments that using well-validated antibodies can save researchers’ time, as well as avoid wasting precious sample material. “Rigorous validation by manufacturers can have an enormous impact on the productivity of scientists,” he says. “If manufacturers do their part well, it makes researchers' jobs easier and ensures more reliable results. Scientists should therefore be careful to choose only well-validated antibodies from a trusted source.”

References

1. Baker M. Reproducibility crisis: Blame it on the antibodies. Nature. 2015;521(7552):274-276

2. Bradbury A, Plückthun A. Reproducibility: Standardize antibodies used in research. Nature. 2015;518(7537):27-29

3. Uhlen M, Bandrowski A, Carr S, et al. A proposal for validation of antibodies. Nat Methods. 2016;13(10):823-827.

4. Baker M. 1,500 scientists lift the lid on reproducibility. Nature. 2016;533(7604):452-454.

5. Voskuil JL. The challenges with the validation of research antibodies. F1000Res. 2017 Feb 17;6:161

6. Norden DM, Navia CT, Sullivan JT, Doranz BJ. The emergence of cell-based protein arrays to test for polyspecific off-target binding of antibody therapeutics. MAbs. 2024;16(1):2393785