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Immunohistochemistry (IHC) is used to visualize antigens in tissue sections by specific antigen-antibody interactions, an advantage over traditionally used staining techniques that can identify only a limited number of proteins and tissue structures. A critical problem for IHC has been the lack of reference standards. Standards and reference materials for IHC would be valuable tools to expedite the acceptance and translation of biomarkers, such as telomerase and p53, from research to clinical use. NIST is developing a reference material for improved quality (reproducibility and accuracy) of biomarker imaging of p53 and telomerase that can be used in a wide range of histological and antibody staining applications.
The lack of quantitative imaging methods and reagents impedes the validation of image-based biomarkers. Improved methods for biomarker quantification and an established traceable value assignment would be useful to customers in a wide range of areas that include clinical diagnostics, diagnostic industries, instrument manufacturers, and academic and government research agencies. Quantifiable measurements of cancer biomarkers, such as p53 and telomerase, are needed for accurate assessment of their use in determining the presence of cancer and its degree of progression.
An SRM kit will be developed using well characterized biomarker proteins (telomerase and p53) immobilized on glass slides at various concentrations. The package will also include fluorescently labeled antibodies used for the detection of these biomarker proteins.
The reference device contains immobilized biomarker proteins on a glass slide, which are characterized in activity and concentration by previously established biochemical and physical methods.
A commercial preparation of human p53 protein that has been characterized by 2-D gel chromatography and mass spectrometry is used. Such preparations may have low purity, but mass spectroscopy determines that they contain the designated biomarker and components that may cross react. We will also use 2-D gel chromatography and MALDI-TOF measurements to characterize telomerase from yeast.
The biomarker proteins are immobilized through the amide linkage onto a (3-aminopropyl)trietoxysilane modified glass surface using previously developed methods. This method has been shown to be robust while maintaining the activity of the original protein. The procedure is reproducible and produces a chemically stable product in high yield.
The activity of the immobilized p53 and telomerase proteins are then characterized using IHC and fluorescence microscopy by comparison to A549 cancer cell lines. The digitized IHC images of cells and reference protein are statistically compared, based on the distribution and intensity of staining and proportion of positively stained cells.
• Developed methods for use in the production of a reference device for IHC imaging of biomarkers.
• Developed candidate reference materials for p53 genetic point mutations and telomerase activity assays.