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Updated: Apr 13, 2026
Synthetic Antigen Controls for Immunohistochemistry
Published on: August 23, 2021
Standardizing Immunohistochemistry: A New Reference Control for Detecting Staining Problems
Seshi R Sompuram1, Kodela Vani1, Brian Tracey2
1Medical Discovery Partners LLC, Boston, Massachusetts (SRS,KV,SAB)
Insights
A novel immunohistochemistry (IHC) control using peptide-coated beads offers standardized breast cancer testing. These "IHControls" ensure reliable detection of human epidermal growth factor receptor 2, estrogen receptor, and progesterone receptor, enhancing diagnostic accuracy.
Area of Science:
- Biotechnology
- Cancer Diagnostics
- Immunohistochemistry
Background:
- Standardized immunohistochemistry (IHC) controls are crucial for accurate breast cancer testing.
- Current tissue-based controls can present challenges in reproducibility and standardization across laboratories.
- Need for a robust control system to validate the analytical performance of IHC assays.
Purpose of the Study:
- To introduce and evaluate a new standardized IHC control system, termed "IHControls", for breast cancer biomarker testing.
- To assess the sensitivity and specificity of IHControls in detecting common issues in IHC staining procedures.
- To compare the performance of IHControls against traditional tissue-based controls.
Main Methods:
- Development of IHControls using formalin-fixed peptide antigens (HER2, ER, PR) covalently attached to glass beads.
- Suspension of antigen-coated beads in a liquid matrix that solidifies on a microscope slide.
- Testing IHControls by simulating various staining problems, including reagent degradation and suboptimal antigen retrieval conditions (time, temperature).
Main Results:
- IHControls demonstrated sensitivity in detecting primary and secondary reagent degradation, comparable to tissue controls.
- The controls effectively identified aberrations in antigen retrieval, mirroring the performance of tissue controls.
- Specificity testing confirmed that each bead reacted only with its corresponding IHC antibody, validating assay specificity.
Conclusions:
- IHControls are capable of verifying the analytical components of immunohistochemical staining, similar to tissue controls.
- The bead-based format allows for bulk preparation, promoting day-to-day reproducibility and inter-laboratory standardization.
- IHControls represent a promising advancement for standardized and reliable breast cancer biomarker assessment.
Abstract:
A new standardized immunohistochemistry (IHC) control for breast cancer testing comprises formalin-fixed human epidermal growth factor receptor 2, estrogen receptor, or progesterone receptor peptide antigens covalently attached to 8-µm glass beads. The antigen-coated beads are suspended in a liquid matrix that hardens upon pipetting onto a glass microscope slide. The antigen-coated beads remain in place through deparaffinization, antigen retrieval, and immunostaining. The intensity of the beads' stain provides feedback regarding the efficacy of both antigen retrieval and immunostaining. As a first report, we tested the sensitivity and specificity of the new IHC controls ("IHControls"). To evaluate sensitivity, various staining problems were simulated. IHControls detected primary and secondary reagent degradation similarly to tissue controls. This first group of IHControls behaved similarly to tissue controls expressing high concentrations of the antigen. The IHControls were also able to detect aberrations in antigen retrieval, as simulated by sub-optimal times or temperatures. Specificity testing revealed that each antigen-coated bead was specific for its cognate IHC test antibody. The data support the conclusion that, like tissue controls, IHControls are capable of verifying the analytic components of an immunohistochemical stain. Unlike tissue controls, IHControls are prepared in large bulk lots, fostering day-to-day reproducibility that can be standardized across laboratories.
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