Bioluminescent Method for Detecting Telomerase Activity

Abstract

Abstract Background: Telomerase is a promising biomarker in cancer diagnosis and therapy. The elongation of telomeric repeats catalyzed by telomerase is accompanied by release of six PPi for each TTAGGG repeat (1 pmol PPi/310 pg telomeric repeats). We developed a novel method to measure telomerase activity by use of an enzymatic luminometric PPi assay (ELIPA). Methods: Extracts of cell lines and tissues were incubated with primer at 30 °C for 30 min. Released PPi was converted to ATP by sulfurylase, and ATP was detected by a luciferase bioluminescence system. The ELIPA results were compared with results obtained with the conventional telomeric repeat amplification (TRAP)-ELISA in 42 lung carcinoma tissues and 27 control tissues without malignancy. Results: The lower detection limits of ELIPA and TRAP-ELISA were 5 and 10 cells, respectively. The within-run imprecision (CV) of ELIPA was ≤12%. When compared with TRAP-ELISA, the correlation coefficient (r) was 0.79. When we used the cutoff value from ROC analysis to distinguish malignant and nonmalignant tissues, the sensitivity and specificity of ELIPA were 83% and 96%, respectively, whereas the sensitivity and specificity of TRAP-ELISA were 71% and 96%, respectively. Conclusion: ELIPA is a simple and sensitive homogeneous method to quantify telomerase activity.