Improved methods and standards for telomerase detection: quantitative histopathology using antibody staining

Abstract

Evaluation of telomerase as an early detection biomarker for cancer has been hindered by a lack of reliable methods and standards for in situ histochemical measurement. Improved histochemical methods for measuring telomerase could expedite the acceptance of telomerase as a biomarker for use in diagnostic and clinical applications. The lack of a crystal structure for telomerase coupled with high variability in the antibodies available for immunohistochemical analysis has led to confusion in the literature regarding the binding specificity of these antibodies. We have developed an automated fluorescence microscopy protocol to assess the specificity of three fluorescently labeled telomerase antibodies and to quantify telomerase in cultured human tumor cells and in human fibroblast cells as a control. Significant differences in staining intensity and distribution were observed. Fluorescence measurements in these cell lines were compared to telomerase measured by the telomerase repeat amplification protocol, reverse transcription-polymerase chain reaction, and flow cytometry. This combination of measurements ensured a more complete quantitation of telomerase levels in each of the cell lines and could also be used as a model for validation of other biomarkers for clinical use.

Keywords::

• automation
• biomarker
• cancer
• flow cytometry
• fluorescence microscopy
• immunohistochemistry
• molecular imaging
• quantitation
• telomerase assay

Acknowledgements

The authors gratefully acknowledge the support of the National Institute of Standards and Technology in funding this work through the NIST Division of Standards Reference Materials. Support from a National Research Council–National Institute of Standards and Technology postdoctoral fellowship (NDG) and a NSF–NIST Summer Undergraduate Research Fellowship (SURF) (AHS) are gratefully acknowledged. We also thank Yan Xiao and Science Applications International Corporation (SAIC) for developing the IgY antibody.

Disclaimer

Certain commercial equipment, instruments and materials are identified in this paper in order to specify an experimental procedure as completely as possible. In no case does the identification of particular equipment or materials imply a recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials, instruments, or equipment are necessarily the best available for the purpose.

Declaration of Interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.