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.
- Characterize commercial preparations of human p53 and yeast telomerase
- Investigate immobilization of p53 and telomerase onto a glass slide
- Characterize performance of immobilized p53 and telomerase on a glass slide.
- Provide a reference material that will improve the quality (reproducibility and accuracy) of biomarker imaging using IHC.
Research activities and technical approach
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.