Advanced Antibody Validation Strategies to Ensure Scientific Reproducibility

Although there are millions of antibodies available commercially, some are better validated than others. Choosing the right antibody and confirming that it works for your application and target is critical for generating reproducible results. This article looks at some of the antibody validation strategies used by trusted vendors and suggests how you can implement these methods in your own lab.

Proper validation is essential for vendors to provide high-quality antibodies

A growing number of publications point toward poorly validated antibodies as major contributors to the reproducibility crisis. For example, a 2008 study by Berglund et al. found that when 5,436 commercial antibodies from 51 different antibody providers were independently validated using western blotting and IHC on tissue microarrays, only half received a pass. Notably, failure rates among vendors ranged from 0 to 100%, suggesting that proper validation and quality control is fundamental to providing high-quality reagents. However, a caveat of this investigation is that many of the antibodies had not been vendor-approved for the IHC application.^1,2

Ensuring antibody specificity begins upstream of validation

When developing antibodies, there are several critical steps for ensuring specificity that should be considered upstream of validation processes. According to Dr. Joanna Porankiewicz Asplund, Customer Support Manager at Agrisera Antibodies, antigen selection is of paramount importance. “If the sequence used to elicit an antibody is not unique to the target of interest, the risk of cross-reactivity is increased,” she says. “For example, if a full-length protein is used for immunization, from a protein family, cross reaction with other family members is very likely.”

Purity is another factor that can influence antibody performance. “When developing our monoclonal antibodies, we use multiple chromatography steps to achieve high purity,” reports Shamim Mohammad, Ph.D., Lead Scientist at ATCC. “These include protein A/G affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and size exclusion chromatography. The purity of the final antibody product is assessed during phase 1 of our two-tiered quality control (QC) process, along with properties such as isotype, concentration, and antigen specificity.”

Polyclonals and monoclonals present different reproducibility considerations

It is worth noting that polyclonal and monoclonal antibodies present different reproducibility considerations. Polyclonals are prone to variability between different batches, making it crucial to validate each batch and reoptimize experimental conditions where required. Monoclonals have greater batch-to-batch consistency than polyclonals, but the hybridoma method can result in genetic drift over time. Mohammad notes that ATCC mitigates against genetic drift by using advanced techniques for authenticating hybridomas and antibodies, which include sequencing of antibody heavy and light chains. “This allows for designing and modifying an antibody’s specificity and affinity for diagnostic and therapeutic applications,” he says.

Antibody validation strategies

One of the most obvious ways of validating an antibody is to perform testing with biologically relevant positive and negative expression systems. These could be samples that are known to endogenously express the target of interest or lack target expression. Alternatively, samples could be generated using specific treatments to induce or inhibit target expression, or could be produced with knockout (KO) or knockdown (KD) methods.

“KO validation involves completely removing the gene encoding the target protein, ensuring that any signal detected by the antibody is specific to the target,” explains Tracey Long Ph.D., Senior Marketing Manager, Catalog Antibodies, Life Science Group at Bio-Rad Laboratories. “This method is highly specific but can be time-consuming and costly. KD validation, on the other hand, reduces the expression of the target protein using RNA interference or similar methods. It is quicker and less expensive than KO but may not completely eliminate the target protein, potentially leading to residual signals.”

Another common validation strategy involves western blot detection of the recombinant protein. However, Porankiewicz Asplund cautions that this can be misleading if the antibody datasheet is not read carefully. “Based on this type of data, researchers might expect to see a very intense band on a western blot, not realizing that it is impossible to achieve this in an endogenous extract, for a target of low abundance,” she says. Protein abundance can be easily checked in PaxDb.

Antibodies may also be validated with cell-based reporter assays, such as those offered by Promega Corporation. “Our cell-based bioassays have bioluminescent reporters driven by genetic elements that indicate signaling through specific pathways,” explains Julia Gilden, Ph.D., Sr. Research Scientist. “We express a target receptor on the surface of a reporter cell and introduce a luciferase reporter gene linked to the signaling pathway of interest. If the antibody engages the receptor and effects signaling through the expected pathway, the reporter cell will produce light dependent on the concentration of antibody that has been applied. These types of bioassays add a new level of functional information, allowing scientists to explore whether an antibody has agonist or antagonist activity, measure its potency relative to other antibodies, and test for Fc effector functions.”

Immunoprecipitation followed by mass spectrometry (IP-MS) is a powerful technique for antibody target verification. “IP-MS allows for the precise identification and quantification of target proteins and their interacting partners,” says Long. “Additionally, IP-MS can identify post-translational modifications, offering insights into protein function and regulation. Overall, by identifying not only the target protein but also the off-targets, IP-MS enhances the accuracy and depth of antibody validation.”

Other types of validation methods include testing with several independent antibodies to the same target, which could include using one antibody for immunoprecipitation and another for western blot detection, and orthogonal validation, which involves comparing antibody-based results with non-antibody-based data (e.g., in situ hybridization or RNA-seq). “At ATCC, we additionally perform epitope mapping, assess neutralizing activity, and run competitive assays for epitope binning during tier two of our QC process,” notes Mohammad.

Validation should be tailored to the application and target

Once you have selected an antibody, you should always confirm that it works in your own experimental setting before proceeding with data-driven research. Use the vendor’s recommended protocol as a starting point for optimization, refer to the literature for guidance with testing your particular sample type, and don’t hesitate to ask for help from the antibody manufacturer. A reliable vendor will be invested in your success and can offer expert advice to keep your project moving.

References

1. Berglund L, Björling E, Oksvold P, et al. A genecentric Human Protein Atlas for expression profiles based on antibodies. Mol Cell Proteomics. 2008;7(10):2019-2027. doi:10.1074/mcp.R800013-MCP200

2. Acharya P, Quinlan A, Neumeister V. The ABCs of finding a good antibody: How to find a good antibody, validate it, and publish meaningful data. F1000Res. 2017;6:851. doi:10.12688/f1000research.11774.1