Bioluminescent method for detecting telomerase activity.
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 PP(i) for each TTAGGG repeat (1 pmol PP(i)/310 pg telomeric repeats). We developed a novel method to measure telomerase activity by use of an enzymatic luminometric PP(i) assay (ELIPA).


METHODS
Extracts of cell lines and tissues were incubated with primer at 30 degrees C for 30 min. Released PP(i) 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 < or =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.


CONCLUSIONS
ELIPA is a simple and sensitive homogeneous method to quantify telomerase activity.
Xu
Shun-Qing
SQ
National Laboratory of Biomedical Photonics, Institute of Environmental Medicine, Tongji Medical College of Huazhong University of Science and Technology, 13 Hangkong Rd., Wuhan 430030, The People's Republic of China. shunqing@public.wh.hb.cn
He
Min
M
Yu
Hong-Ping
HP
Wang
Xiao-Yang
XY
Tan
Xiang-Lin
XL
Lu
Bin
B
Sun
Xi
X
Zhou
Yi-Kai
YK
Yao
Qun-Feng
QF
Xu
Yong-Jun
YJ
Zhang
Zhi-Ren
ZR
D000255:Adenosine Triphosphate / Q000737:chemistry; D014408:Biomarkers, Tumor / Q000032:analysis*; D002384:Catalysis; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008156:Luciferases; D008163:Luminescent Measurements; D008168:Lung / Q000737:chemistry; D008175:Lung Neoplasms / Q000737:chemistry / Q000175:diagnosis; D012091:Repetitive Sequences, Nucleic Acid; D015203:Reproducibility of Results; D019098:Telomerase / Q000032:analysis*; D014407:Tumor Cells, Cultured
D014408:Biomarkers, Tumor; D000255:Adenosine Triphosphate; D008156:Luciferases; D019098:Telomerase