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Updated: Oct 21, 2025
Synthetic Antigen Controls for Immunohistochemistry
Published on: August 23, 2021
Synthetic Antigen Controls for Immunohistochemistry
Charles A Havnar1, Kathy J Hötzel1, Charles A Jones1
1Research Pathology, Genentech, Inc.
Insights
A novel method creates synthetic immunohistochemistry (IHC) controls using known antigen concentrations. This approach ensures reliable protein expression analysis when traditional controls are unavailable, improving assay validation.
Area of Science:
- Biotechnology
- Histology
- Immunology
Background:
- Immunohistochemistry (IHC) relies on accurate positive and negative controls for interpreting protein expression.
- Availability of suitable tissue or cell line controls for IHC can be limited.
Purpose of the Study:
- To present a simple, adaptable protocol for generating synthetic immunohistochemistry (IHC) controls.
- To enable creation of controls for various antigen types and concentrations.
Main Methods:
- Synthetic controls were created using serial dilutions of a human erythroblastic oncogene B2 (ERBB2/HER2) intracellular domain (ICD) peptide in bovine serum albumin (BSA).
- The peptide/BSA mixture was cross-linked with formaldehyde and heated, then processed, sectioned, and stained similarly to tissue samples.
Main Results:
- The protocol yields a series of samples with known antigen concentrations, demonstrating a wide range of staining intensities.
- The method is compatible with routine histology laboratory procedures.
Conclusions:
- This technique provides a straightforward way to generate well-characterized synthetic IHC controls when endogenous controls are lacking.
- The ability to create controls with defined antigen levels aids in assessing assay performance, dynamic range, and optimization.
Abstract:
Immunohistochemistry (IHC) assays provide valuable insights into protein expression patterns, the reliable interpretation of which requires well-characterized positive and negative control samples. Because appropriate tissue or cell line controls are not always available, a simple method to create synthetic IHC controls may be beneficial. Such a method is described here. It is adaptable to various antigen types, including proteins, peptides, or oligonucleotides, in a wide range of concentrations. This protocol explains the steps necessary to create synthetic antigen controls, using as an example a peptide from the human erythroblastic oncogene B2 (ERBB2/HER2) intracellular domain (ICD) recognized by a variety of diagnostically relevant antibodies. Serial dilutions of the HER2 ICD peptide in bovine serum albumin (BSA) solution are mixed with formaldehyde and heated for 10 min at 85 °C to solidify and cross-link the peptide/BSA mixture. The resulting gel can be processed, sectioned, and stained like a tissue, yielding a series of samples of known antigen concentrations spanning a wide range of staining intensities. This simple protocol is consistent with routine histology lab procedures. The method requires only that the user have a sufficient quantity of the desired antigen. Recombinant proteins, protein domains, or linear peptides that encode relevant epitopes may be synthesized locally or commercially. Laboratories generating in-house antibodies can reserve aliquots of the immunizing antigen as the synthetic control target. The opportunity to create well-defined positive controls across a wide range of concentrations allows users to assess intra- and inter-laboratory assay performance, gain insight into the dynamic range and linearity of their assays, and optimize assay conditions for their particular experimental goals.
