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Validating Immunohistochemistry Assay Specificity in Investigative Studies: Considerations for a Weight of Evidence
Joshua D Webster1, Margaret Solon1, Katherine N Gibson-Corley2
17412Genentech, South San Francisco, CA, USA.
Insights
Immunohistochemistry (IHC) validation is crucial for reliable protein expression analysis. This review explores various methods to confirm antibody specificity, ensuring accurate interpretation of IHC results in research and diagnostics.
Area of Science:
- Pathology
- Molecular Biology
- Immunology
Background:
- Immunohistochemistry (IHC) is a key technique for visualizing protein expression within tissue context.
- Accurate IHC interpretation relies heavily on validated antibody specificity.
Purpose of the Study:
- To review diverse approaches for validating immunohistochemistry (IHC) antibody specificity.
- To emphasize the importance of antibody validation in investigative studies.
Main Methods:
- Discussion of validation strategies including bioinformatics, tissue controls, cell pellets, and orthogonal methods.
- Analysis of the strengths and weaknesses of each validation approach.
- Emphasis on a cumulative "weight of evidence" for antibody specificity.
Main Results:
- Multiple validation methods exist, each with limitations.
- A combination of approaches provides stronger evidence for specificity.
- Assessing validation impacts the interpretation of IHC data.
Conclusions:
- Achieving absolute confidence in IHC specificity is challenging.
- Thoughtful consideration of validation evidence is essential for reliable IHC results.
- This review aims to stimulate critical thinking regarding IHC antibody validation and interpretation.
Abstract:
Immunohistochemistry (IHC) is a fundamental molecular technique that provides information on protein expression in the context of spatial localization and tissue morphology. IHC is used in all facets of pathology from identifying infectious agents or characterizing tumors in diagnostics, to characterizing cellular and molecular processes in investigative and experimental studies. Confidence in an IHC assay is primarily driven by the degree to which it is validated. There are many approaches to validate an IHC assay's specificity including bioinformatics approaches using published protein sequences, careful design of positive and negative tissue controls, use of cell pellets with known target protein expression, corroboration of IHC findings with western blots and other analytical methods, and replacement of the primary antibody with an appropriate negative control reagent. Each approach has inherent strengths and weaknesses, and the thoughtful use of these approaches provides cumulative evidence, or a weight of evidence, to support the IHC assay's specificity and build confidence in a study's conclusions. Although it is difficult to be 100% confident in the specificity of any IHC assay, it is important to consider how validation approaches provide evidence to support or to question the specificity of labeling, and how that evidence affects the overall interpretation of a study's results. In this review, we discuss different approaches for IHC antibody validation, with an emphasis on the characterization of antibody specificity in investigative studies. While this review is not prescriptive, it is hoped that it will be thought provoking when considering the interpretation of IHC results.
