Antibody Enhanced Validation – Additional Validation Data to Help Ensure Specificity and Performance

Reproducibility in biological systems

One of the assumptions on which scientific progress is founded is that published results may be recapitulated by any trained scientist using the published method and materials. The life sciences are currently challenged by what some have called a crisis of reproducibility, some of which centers on the consistent identity and application of reagents----particularly antibodies, which are produced in living organisms and therefore have the potential for variability. These attributes have led some researchers to question the reproducibility of antibodies for research and clinical applications.

The importance of antibody validation

Supplier characterization of antibody binding in relevant research applications and appropriate researcher selection and optimization is essential for confidence in purchase of an antibody product and the scientific conclusions that may follow from results of their use. Subsequent experimental work based on erroneous or misinterpreted immunodetection results poses significant risk that such results could lead to extensive misapplication of time, effort, and expense—and misdirected conclusions that have the potential to impact human lives.

Current commercial antibody validation practices

It is common for commercial antibody developers to assess the on-target binding of an antibody during post immunization screening solely by Western blot (WB) or immunohistochemistry (IHC). This screen ensures that the antibody in development is, at this initial stage, recognizing the expected target and performing similarly to previous lots, if applicable. However, this simple screening is not adequate to assure application suitability and true lot-to-lot consistency.

We therefore subsequently test in as many additional immunodetection applications as practical in samples chosen to be relevant to the intended use of the product. These include immunohistochemistry, immunocytochemistry(ICC), Western blot, ELISA, immunoprecipitation, and more. This in-depth application testing can help assess the antibody’s specificity for the target and provides contextually relevant validation in applications and samples most likely to be used by our customers. This application-specific data should be reviewed by the researcher and appropriately assessed for the researchers intended use. Beyond review of the vendor generated application data, critical review of the epitope, species reactivity, clonality, appropriate host species, and development of appropriate controls are critical responsibilities of the researcher in the selection and use of antibody product.

Western blot analysis using anti-ATRX antibody in human cell line A-549. Cat. No. MABT118

 Immunohistochemistry validation of anti-desmoglein antibody using human tonsil tissue


Immunohistochemistry validation of anti-desmoglein antibody using human tonsil tissue. Membrane junction immunoreactivity was observed in the stratified squamous epithelium (A) as well as the epithelial cells lining the crypts of the human tonsil (C). Treatment of the same tissues with negative control reagent (B, D) resulted in no detectable signal.
Cat. No. MABT118

The proposed ‘enhanced’ solution

Many concerned scientists have called for more rigorous evidence that commercial antibodies specifically bind the intended target. The International Working Group for Antibody Validation (IWGAV) has recommended best approaches for validating antibodies used in common research applications, and has published suggested guidelines that are intended to enhance antibody reproducibility. The IWGAV has suggested ‘pillars’ for validation of antibodies that would enhance confidence in their function in biomedical and immunodetection applications. 

Our enhanced validation efforts

In answer to calls for more stringent corroboration of immunodetection application-based antibody validation, we are pleased to offer, enhanced validation for many antibodies in our portfolio. Enhanced validation (EV) data that are now represented on our product pages align with several of the 2016 IWGAV guidelines for more rigorous specificity testing, and are available for those antibodies in our catalog that carry the EV symbol. All antibodies we offer that carry the ‘EV’ symbol will be accompanied by data resulting from one or more of these enhanced validation strategies.

Genetic Strategies

Genetic Strategies – Demonstrating antibody specificity through knockout/knockdown methods

Expected Results: Diminished or absence of band in Western blotting in knockdown/knockout validation.

The genetic strategies pillar attempts to ensure specificity of protein detection by eliminating or reducing the expression of the target protein through gene editing or gene interference methods and comparing the antibody recognition for such a sample compared to the wild type. The specificity of the antibody can subsequently be assessed through WB or ICC staining of the cell sample that has been altered to reduce or eliminate the target protein expression, and compared to the wild type sample that has been demonstrated to express the target protein.

CRISPR/Cas9 genome editing technology enables the creation of cell lines with a specific gene or genes permanently excised so that expression of the corresponding protein is ‘knocked out’. For antibody validation this poses a potentially powerful tool. In simplest terms, a sample with the target “knocked out” should show no antibody staining as the intended target has been genetically removed and the corresponding wild type sample should demonstrate recognition of the target. Degrees of non-specificity can be assessed by comparing these two sample types.

Alternatively, RNA interference (RNAi) can be used to “knock down” or suppress a specific gene to obtain a reduced protein expression. As with the knockout approach, one would compare the genetically altered “knockdown” sample to the wild type. In contrast with genetic knockout, antibody reactivity signal observed in RNAi-altered samples should be reduced in intensity compared to the wild type and often not completely abolished.





Example of genetic validation by RNAi knockdown. Western blot analysis using an anti-ATRX antibody in A-549 cells transfected with control siRNA and two target-specific siRNA probes. Downregulation of ATRX in siRNA samples confirms specificity of antibody.


Independent Antibody Verification

Independent Antibody Verification – Demonstrating antibody specificity through the use of multiple antibodies against target in IHC or ICC.

Expected results: All antibodies should show similar staining patterns or experimental results.

The validity of results obtained with an antibody in a given immunoapplication may be supported by showing that the same results are obtained using the identical protocol with a different antibody raised against the same target. At least two antibodies with non-overlapping epitopes are applied across a panel of samples, such as sections from the same tissue. This approach has the added advantage of enabling validation of both antibodies used for comparison of binding characteristics.

  HPA038847, anti-A2ML1 antibody HPA038848, different anti-A2ML1 antibody
  Immunohistochemical staining using an anti-A2ML1 antibody of human esophagus shows high expression. Two anti-A2ML1 antibodies HPA038847 (left) and HPA038848 (right) target different regions of A2ML1. Staining across positive and negative tissues show similar staining profiles between the two antibodies, demonstrating Independent Antibody Verification. Positive staining for A2ML1 is shown using esophagus tissue, bottom (known to express A2ML1) and negative staining is shown using colon tissue, top (A2ML1 not expressed).


Orthogonal Validation Using RNA-seq

Orthogonal Validation Using RNA-seq - Demonstrating antibody specificity through an antibody-dependent method correlated with an antibody-independent method (RNA-seq).

Expected Results: Antibody staining intensities should correlate to RNA-seq data from the same samples.

In the antibody validation process, the practice of orthogonal validation refers to the comparison of immunodetection results with a method that is antibody-independent for measuring the gene or protein of interest.

The orthogonal technique, RNA-seq, is a method used to quantify gene expression at the mRNA level for a given sample for a specific target. This expression at the mRNA level should correspond to protein expression. For this pillar of enhanced antibody validation, data is presented demonstrating the mRNA expression level obtained by RNA-seq for a given target in bar graph form for a high- and a low-expressing tissue (5-fold difference) alongside WB or IHC data for those same samples. What is demonstrated is a direct comparison of mRNA expression level and sample staining: High expression=strong staining; Low expression=weak or no staining.

Orthogonal validation using RNAseq.


Orthogonal validation in Western blot (left) and IHC (right). Samples with known high and low RNA expression were chosen. Left: Western blot analysis (left) using an anti-VIM antibody in human cell lines U-251MG (high expression of VIM) and MCF-7 (low expression of VIM) correlates with RNA-seq data (right).
Right: Immunohistochemistry analysis using an anti-PLA2R1 antibody in human kidney (left, high expression of PLA2R1) and pancreas tissues (right, low expression of PLA2R1) correlates with RNA-seq data.

Functional Assay Validation

Functional Assay Validation - Demonstrating antibody specificity through immunodetection of experimentally induced changes in target antigen expression and or activity levels.

Expected Results: Immunodetection of altered antigen activity or expression levels via WB, ICC, IF, and FS.

Many antigen expression and activity levels can be readably modulated in vitro by experimental manipulation. Leveraging common protein expression or bioactivity-altering techniques allows for the determination of antibody specificity by detecting changes in the activation or expression levels of the target protein. Functional assay validation is a valuable EV pillar as it provides strong supporting evidence of antibody specificity.



For the functional assay validation pillar, HeLa cells were incubated in EBSS medium for 2 hr and autophagosome staining is demonstrated in the experimentally induced HeLa cells (A) while no autophagosome staining is demonstrated in the untreated cells (B). Cells were fixed and permeabilized with cold methanol followed by cold acetone and stained with 5 μg/mL Rabbit Anti-LC3B (Cat. No. L7543), a marker for autophagosome membranes. The antibody was developed using Goat Anti-Rabbit IgG, Cy3 conjugate.

Our steadfast commitment to antibodies validated for life science research

We are a leading provider of primary antibody products that are validated every day by your peers through the immense number of peer reviewed citations of the use of our antibodies. Industry leading quality control processes and application testing ensure that you can rely on our antibodies to provide the expected performance the first time and every time. In addition to our long-standing commitment to validation, we continue to expand and improve our processes by incorporating more rigorous validation strategies into the development of many of our antibody reagents. We stand by the quality of our product with our Antibody Guarantee—if our antibody does not perform as specified in your application, we will issue a full credit or replacement antibody product.

General References

  • Baker, Monya. Blame it on the antibodies. Nature. 2015 May;521:274-6.
  • Uhlen, M., Bandrowski A., Carr, S., et al. A proposal for validation of antibodies. Nat Meth. 2016 Oct; 13(10): 823-27.
  • Unniyampurath, U., Pilankatta, R., Krishnan, M.N. RNA interference in the age of CRISPR: will CRISPR interefere with RNAi? Int J Mol Sci. 2016 Mar; 17(3): 291-316.
  • Wang, Z., Gerstein, M., Snyder, M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009 Jan;10(1):57-63.


For Research Use Only. Not for use in diagnostic procedures.

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.