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The Importance of Antibody Validation

One of the most critical steps in multiplexed antibody panel design is selecting the right antibodies for your study. Choose the wrong antibody and you may lose weeks if not months, worth of work. A rigorous antibody validation strategy keeps your program on track by:

  1. Proving antibody specificity (the antibody’s ability to differentiate between various antigens)
  2. Proving affinity (antibody-epitope binding intensity)
  3. Demonstrating reproducibility

We asked our frequent collaborators at Cell Signaling Technology (CST) how they test and validate specificity of immunological reagents. Here’s what we learned.

Antibody validation is not a one-size-fits-all process. There is no single assay that can determine the validity of an antibody. To ensure their antibodies will work reliably across a broad range of experiments, CST developed their Hallmarks of Antibody Validation™ approach, which is based on six complementary strategies they use to determine antibody functionality, specificity, and sensitivity.

Antibody Validation Wheel

CST Hallmarks of Antibody Validation

Binary Strategy. One of the best ways to evaluate antibody specificity is to test an antibody in biologically relevant positive and negative expression systems. Binary models may include endogenous cells or tissues in which expression of the target protein is known or predicted to be positive or negative, as well as genetic knockouts, or use of treatments to induce or inhibit expression. CST recommends that binary expression data should always be verified using an orthogonal method, such as genetic sequencing. They caution that just because an antibody is specific by western blot doesn’t mean you’ll see the same results with immunohistochemistry (IHC).

Ranged Strategy. By utilizing both endogenous and heterologous models that express high, moderate, and low levels of the target of interest, a ranged approach helps reveal optimal working conditions for a given antibody. Ranged models rely on differences in target expression or modification that are not black and white –and are typically more reflective of real biology. Typical methods include siRNA and heterozygous knockout assays.

“Antibody validation is not a one-size-fits-all process. There is no single assay that can determine the validity of an antibody.”

Orthogonal Strategy. Cross-referencing antibody results using data from non-antibody-based methods and resources will not only enable you to verify existing antibody validation data, but also to identify any effects or artifacts directly related to your antibody of interest. Orthogonal approaches include evaluating previously published results, studying expression analysis using various ’omics techniques (e.g., genomics, transcriptomics, or proteomics), performing in situ hybridization, RNA sequencing, and mass spectrometry.

Multiple Antibody Strategy. One of the most powerful tools for antibody validation, this approach compares the signal of the antibody of interest to that of antibodies targeting non-overlapping epitopes of the desired target. A common method is to first immunoprecipitate (IP) the target with one antibody, then subsequently detect it by western blot, using another antibody against the same target. This helps verify that both antibodies bind to the correct molecule. In addition to IP, ChIP and ChIP-seq are commonly used methods for the multiple antibody strategy.

Heterologous Strategy. This approach involves evaluating the antibody signal in cell lines following heterologous expression of a native or mutated target protein. Useful for verifying cross-reactivity of an antibody with protein isoforms or conserved family members, it can also reveal potential for off-target binding due to antigen homology. Recombinant proteins may be used to identify specificity of an antibody for one or more members of a protein family. Alternatively, DNA plasmids designed for exogenous expression of antigenic targets of interest can be analyzed by western blot or immunocytochemical staining techniques.

Complementary Strategies. Another method for validating antibody specificity is to use complementary assays, such as competitive ELISA, peptide dot blots, peptide blocking, or protein arrays. These techniques can provide important information about antibody specificity and functionality. In addition, they may be tailored to the biological nature of the target as well as to fit requirements of downstream assays.

To learn more, visit CST Antibody Validation Principles. You may also want to read the 2016 Nature Methods paper by Uhlen et al. on this topic: “A proposal for the Validation of Antibodies”.  CST adapted recommendations in this paper to develop their proven validation strategies.

Ab validation in multiple tissue types 01

CST used cancer tissue microarrays to assess antibody performance in non-Hodgkin’s lymphoma, colon carcinoma, and serous carcinoma of ovary (left to right) tissue samples from mouse models. These tissues were stained for CD163 (D6U1J), a phenotypic marker of M2 macrophages, using Rabbit mAb (CST #93498).

Antibody Validation for Immunohistochemistry

To ensure IHC studies yield accurate, reproducible results, CST follows validation protocols that ensure that the staining you observe with each CST antibody is specific and reliable.  CST scientists employ a variety of methods to:

  • Identify presence of desired protein molecules using western blot analysis that demonstrates bands of the appropriate weight with minimal cross-reacting bands
  • Verify target specificity using paraffin-embedded cell pellets of known target expression levels
  • Assess antibody performance in relevant mouse models
  • Further verify target specificity by generating xenografts from cell lines with known target expression levels
  • Demonstrate antibody performance over a broad spectrum of tissue types using human tissue arrays
  • Verify target phospho-specificity by subjecting tissue sections and cell pellets to phosphatase treatment
  • Verify antibody specificity and rule out Fc-mediating binding by using blocking peptides

For more tips on validating antibodies and other factors to consider when designing multiplex antibody panels, watch our Akoya Academy webinar on Best Practices for Developing Custom CODEX Panels.

Hallmarks of Antibody Validation is a trademark of Cell Signaling Technology, Inc.

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