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Abstract: TH-PO574

Modulation of Linear Ranges in Immunohistochemistry and Immunofluorescence Assays

Session Information

  • Pathology and Lab Medicine
    November 03, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
    Abstract Time: 10:00 AM - 12:00 PM

Category: Pathology and Lab Medicine

  • 1700 Pathology and Lab Medicine


  • Ferkowicz, Michael J., Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Sompuram, Seshi R., Boston Cell Standards, Boston, Massachusetts, United States
  • Barwinska, Daria, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • El-Achkar, Tarek M., Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Bogen, Steve, Boston Cell Standards, Boston, Massachusetts, United States

Group or Team Name

  • KPMP

Assays for the detection of specific proteins in tissue sections are usually performed by immunohistochemistry (IHC) or immunofluorescence (IF). These are qualitative assays, indicating whether the protein is detectable or undetectable. Attempts have been reported to express relative quantification of protein analytes (in IHC or IF) by describing the staining as weak, moderate, strong, or as percent of positive cells. However, the actual linear range of these assessments has not been determined. The ability to determine the quality of an antibody or lot over time is crucial. Connecting the presence of staining to cellular protein concentrations has never been possible.
We hypothesize that using IHC/IF calibrators, newly developed by our group, it will be feasible to characterize linear ranges of IHC and IF assays. By adjusting the reagents or staining protocol, it is possible to adjust the linear ranges for greater or lesser analytic sensitivity.


Four calibrator targets (analytes), developed for the Kidney Precision Medicine Project (KPMP) - Aquaporin-1 (AQP1), Uromodulin (UMOD), Myeloperoxidase (MPO) and CD3 were covalently attached to glass microbeads across a range of concentrations simulating different cellular expression levels. The cell-sized microbeads were then adhered to microscope slides, stained, and imaged by confocal microscopy. Mean fluorescence intensity of microbeads across ten concentrations was measured.


Assessment of mean fluorescent intensity revealed that all four assays have a linear range of detection when measured by either IF or IHC. Furthermore, we observed that IF can demonstrate a broader linear range due to the inherent ability to tune the confocal microscope.


IHC/IF calibrators are a useful tool for assessing antibody quality, lot to lot consistency, and degradation over time. These data also establish a foundation for using IHC and IF assays for quantifying cellular expression levels of proteins in tissue sections.


  • NIDDK Support