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Updated: Feb 27, 2026
Synthetic Antigen Controls for Immunohistochemistry
Published on: August 23, 2021
The Importance of Epitope Density in Selecting a Sensitive Positive IHC Control
Kodela Vani1, Seshi R Sompuram1, Anika K Schaedle1
1Medical Discovery Partners LLC, Boston, Massachusetts (KV, SRS, AKS, AB, SAB).
Insights
Optimizing immunohistochemistry (IHC) controls is crucial for accurate results. Using well-defined analyte concentrations in positive IHC controls, rather than strong staining ones, enhances sensitivity for detecting reagent degradation.
Area of Science:
- Biotechnology
- Pathology
- Laboratory Medicine
Background:
- Clinical immunohistochemistry (IHC) laboratories require accurate and reproducible immunostains.
- Homemade positive controls from pathological samples often have unknown epitope density, impacting control performance.
- The effect of defined analyte concentrations on IHC control sensitivity remains unclear.
Purpose of the Study:
- To prepare positive IHC controls (IHControls) with defined, homogeneous, and reproducible analyte concentrations for HER-2, ER, and PR.
- To investigate the impact of analyte concentration on the sensitivity of IHC controls in detecting reagent degradation.
Main Methods:
- Preparation of IHControls with precisely quantified analyte concentrations for HER-2, ER, and PR.
- Evaluation of IHControls and conventional tissue controls in simulated primary antibody reagent degradation experiments.
- Analysis of the relationship between control analyte concentration and immunostain analytic response curves.
Main Results:
- The sensitivity of a positive IHC control in detecting reagent degradation is contingent upon its analyte concentration and its position on the immunostain's analytic response curve.
- Positive IHC controls with analyte concentrations near or within the concentration-dependent response range exhibit the highest sensitivity.
- Strongly staining positive controls, with analyte concentrations on the plateau of the response curve, are less sensitive to reagent degradation.
Conclusions:
- Selecting positive IHC controls with intermediate staining intensity is more effective for revealing reagent degradation than those with strong intensity.
- Defined analyte concentrations in positive IHC controls are critical for ensuring reliable and sensitive detection of immunostain performance issues.
- This study highlights the importance of optimizing positive control characteristics for improved diagnostic accuracy in clinical IHC.
Abstract:
Clinical Immunohistochemistry (IHC) laboratories face unique challenges in performing accurate and reproducible immunostains. Among these challenges is the use of homemade controls derived from pathological discard samples. Such positive controls have an unknown number of analyte molecules per cell (epitope density). It is unclear how the lack of defined analyte concentrations affects performance of the control. To address this question, we prepared positive IHC controls ( IHControls) for human epidermal growth factor receptor type II (HER-2), estrogen receptor (ER), or progesterone receptor (PR) with well-defined, homogeneous, and reproducible analyte concentrations. Using the IHControls, we examined the effect of analyte concentration on IHC control sensitivity. IHControls and conventional tissue controls were evaluated in a series of simulated primary antibody reagent degradation experiments. The data demonstrate that the ability of a positive IHC control to reveal reagent degradation depends on (1) the analyte concentration in the control and (2) where that concentration falls on the immunostain's analytic response curve. The most sensitive positive IHC controls have analyte concentrations within or close to the immunostain's concentration-dependent response range. Strongly staining positive controls having analyte concentrations on the analytic response curve plateau are less sensitive. These findings emphasize the importance of selecting positive IHC controls that are of intermediate (rather than strong) stain intensity.
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