Validating your cancer biomarker antibody for IHC

Correct validation of your IHC antibody is essential for accurate biomarker analysis. 

Download the complete Cancer biomarkers guide here. 

Updated July 6, 2023

Immunohistochemistry determines the cellular or subcellular localization of a specific protein. This technique plays a vital role in diagnostics and is increasingly used to target drug therapies as a quick and cost-effective assay^1,2. Biomarkers for conditions such as cancer are commonly analyzed via IHC to yield essential diagnostic and prognostic information and to monitor treatments.

Interpreting positive and negative signals in IHC assays can be difficult. In addition to the acknowledged problems of antigen retrieval in paraffin-embedded sections, the differential availability of antigens between different assay formats means that an antibody recognizing a single band on a western blot may recognize multiple proteins in IHC.

There is no easy way around these problems. The usual approach is to compare samples to both positive and negative control sample staining and to demonstrate that similar signals can be obtained using multiple antibodies against the same target.

A crucial step prior to IHC analysis is antibody validation; this will ensure your antibody is specific to the cancer biomarker of interest and sufficiently sensitive to allow IHC analysis over the required dynamic range demanded by the pathology.

Early validation of your cancer biomarker antibody offers confidence in your results and allows a better understanding of the target. Work produced with validated antibodies ensures long-term reproducibility of results and is quicker to transition to the clinical setting³.

At abcam, we perform rigorous antibody validation for IHC to guarantee antibody specificity, sensitivity and reproducibility. We include numerous reviews direct from researchers highlighting how our antibodies perform hands-on across many different labs.

Important IHC antibody validation steps

No cross-reactivity: Determine cross-reactivity and target specificity in an application such as western blotting⁴. This allows easy visualization of your antibody specificity.

Anti-ERG antibody [EPR3864] (ab92513) validation. Western blot analysis shows ERG at the expected mol weight of 55kDa with no cross-reactivity in Jurkat cells (lane 1) and HeLa cells (lane 2).

Correct IHC staining pattern: Use both positive and negative expressing tissue samples with known localization patterns to confirm the antibody still specifically and sensitively binds the target following formalin fixation and antigen retrieval processes.

Confirm testing in relevant tissue: Antibody testing in tissue samples that are representative of the end-use is important. This includes testing across healthy and related diseased tissues to confirm antibody specificity and sensitivity in your tissue of interest.

^{Figure: IHC staining of ERG in FFPE tissue samples (a) Human kidney tissue with negative control inset (b) mouse brain tissue (c) human prostate cancer (d) human brain tissue with Anti-ERG primary antibody (ab92513) show the expected protein localization in each tissue type.}

The antibody is only considered specific if it repeatedly demonstrates the expected localization across all positive and negative controls.

Reproducible antibody performance: Consistent antibody performance is critical to the long-term viability of research results. Recombinant antibodies are defined by their sequence, so can be reproducibly manufactured with the least batch-to-batch variation among affinity reagents⁵.

Abcam recombinant antibodies offer excellent target specificity, sensitivity, and reproducibility.

Validating reproducibility in IHC antibodies requires comparison for each new batch of antibody produced, to determine batch-to-batch consistency with reproducible, clear staining of the target protein in each of the relevant tissues.

^{Figure: Batch-to-batch reproducibility in the localization of PD-L1 in FFPE human placenta across five batches of Anti-PD-L1 antibody [28-8] (ab205921). IHC performed manually using a Biocare Decloaking Device and Universal HIER antigen retrieval reagent (ab208572). The primary antibody was incubated overnight at 4°C, followed by Goat anti-rabbit secondary antibody and HRP-linked anti-goat polymer antibody (ab209101). DAB was used as a chromogen with a hematoxylin counterstain.}

Validating in your lab

We recommend that you perform initial in-house validation with your cancer biomarker antibody before you begin your experiments.

• For well-established IHC antibodies, your own in-house validation can be as straightforward as reproducing the expected result for your cancer biomarker antibody on positive and negative tissues to gain an appropriate signal².
• When investigating validated cancer biomarkers in a new tissue or sample preparation, or novel cancer biomarkers, your own validation may include positive control material from previously investigated tissue. Antibody staining and signal consistency should match those from previously validated tissues. This allows you to optimize your antibody for use in your sample of interest².  

References

1. Yu J, Kane S, Wu J, et al. Clin. Cancer Res. 2009;15:3023–3028. 2.
2. Howat WJ, Lewis A, Jones P, et al. Methods. 2014; 70(1):34-38.
3. Deutsch EW, Ball CA, Berman JJ, et al. Nat. Biotechnol. 2008;26:305–312.
4. Schuster C, Malinowsky K, Liebmann S, et al. Histopathology. 2012;60:E37–E50. 5.
5. Bradbury, A. & Plückthun, A. Nature 2015;518, 27-29.