Case Study: Customized IHC Antibody Validation Services for Antibody Manufacturers

CASE STUDY

Cancer R&D researchers have adopted the use of immunohistochemistry (IHC) tests for research, prognostic and diagnostic use at an extremely fast rate (1). Despite these statistics, many antibody vendors targeting these researchers do not validate whether or not their antibodies can be employed in an IHC test. This is of concern since antibodies can sometimes bind differently depending on the assay that is used (2). In fact, in a Nature Feature entitled, “Reproducibility crisis: Blame it on the Antibodies”, it was shown that less than 50% of commercially available antibodies can be effectively used for IHC tests (3). To address this need, Reveal Biosciences has developed a comprehensive and customizable IHC and immunofluorescence (IF) antibody validation service specific for our antibody vendor clients that allows them to differentiate themselves in this highly commoditized market.

Here we present a typical antibody validation study to illustrate how this service is performed. This service is fully customizable to fit your requirements and budget.

METHOD

Samples

  • Samples may be formalin fixed paraffin embedded (FFPE) or frozen tissue from human, mouse or rat. Reactivity to multiple species could be tested as necessary. Tissues are selected based on known expression profiles determined from published literature or online resources such as the Human Protein Atlas (proteinatlas.org). Reveal can provide or source tissue as needed.

IHC Procedure (IF procedure available on request)

  1. Samples are sectioned at 4um onto positively charged slides.
  2. Immunohistochemistry is performed on a Leica Bond automated immunostainer. Heat-induced antigen retrieval (HIER) is performed as necessary using Leica Bond Epitope Retrieval Buffer 1 (citrate buffer, pH 6.0) for 20 minutes. Leica Bond Epitope Retrieval Buffer 2 (EDTA buffer, pH 9.0) may also be tested on request.
  3. Primary antibodies are tested at 4 different concentrations on positive control tissue to determine the optimal staining conditions. The number of antibody concentrations and antigen retrieval conditions tested can be customized depending on your needs.
  4. Novocastra Bond Refine Polymer Detection secondary reagents are used to detect the primary antibodies. Antibody vendors may also provide secondary antibodies as an alternative to the Leica reagents if required. Secondary antibodies are visualized using 3,3-Diaminobenzidine (DAB – brown). Hematoxylin (blue) is also applied to serve as a nuclear counterstain.
  5. For negative controls, an isotype control is used instead of the protein-specific primary antibody.
  6. Optimal staining conditions are selected by the experienced IHC scientists at Reveal and reported to the client together with the staining protocol used.
  7. Whole slide images are generated in bright field for the optimal staining conditions selected using a Panoramic SCAN (3D Histech). Positive and negative high resolution single field images are provided for each antibody tested.
  8. A Board Certified Pathologist is available to provide descriptions of the protein localization as required.

RESULTS

Figure 1. Representative images of antibody immunostaining on FFPE human tonsil samples. A negative control was performed in the absence of primary antibody (left). Positive immunoreactivity (right) was visualized with DAB (brown). Hematoxylin was used as nuclear counterstain (blue). The scale bar represents 20 µm.
Table 1. Sample Table: Optimal Antibody Concentration in Blue

Deliverables

Antibody validation report containing:

  • High resolution snapshot of IHC or IF staining for each antibody plus negative control
  • A detailed protocol including optimal staining conditions for each antibody

CONCLUSION

Antibody validation services for antibody manufacturers facilitates testing and confirmation of which of the vendor provided antibodies can be used in IHC and IF. Moreover, for each antibody we generate a detailed protocol that can be provided to end users. Quality IHC or IF images are obtained and can be used in marketing materials. Outstanding antibodies can also be added to our list of optimized antibodies upon request, and offered to our biopharma clients for use in future studies.

In conclusion, this comprehensive and customized antibody validation service allows our antibody vendor clients to outperform their competitors and position their products to address the rapidly growing IHC and IF market.

REFERENCES

  1. Grand View Research, Immunohistochemistry (IHC) Market Analysis By Product (Antibodies, Reagents, Equipment, Kits), By Application (Diagnostics, Drug Testing), By End-Use (Hospitals, Diagnostic Laboratories), & Segment Forecasts, 2018 – 2025.
  2. Saper CB, A Guide to the Perplexed on the Specificity of Antibodies, J Histochem Cytochem 57:1–5, 2009.
  3. Baker, M, Reproducibility crisis: Blame it on the antibodies, Nature. 2015 May 21; 521(7552).