Development of a quantitative luminometric hybridization assay for the determination of telomerase activity.
OBJECTIVE
To develop a quantitative luminometric hybridization assay for the determination of telomerase activity in tissue and cell extracts.


METHODS
Quantification is based on the coamplification of telomeric repeats synthesized by telomerase along with a specifically designed recombinant DNA-internal standard (DNA-IS). The DNA-IS has a similar size and the same primer recognition sites as the telomerase DNA products and differs from them only in a central 18 bp sequence. PCR products are captured on microtiter wells via the biotin-streptavidin system and hybridized with two distinct digoxigenin-labeled oligonucleotide probes that are designed to recognize specifically telomerase products and DNA-IS. The hybrids are quantified by a luminometric reaction using an antidigoxigenin antibody conjugated to alkaline phosphatase. The hybridization assay was validated with the MCF-7 breast carcinoma and leukemia K-562 cell lines and a synthetic telomerase product (R(8)).


RESULTS
Luminescence ratios for telomerase products and DNA-IS were linearly related to the concentration of the pre-PCR product synthesized by telomerase (R(8)), in the range of 0.0005 to 10 pM. The overall reproducibility of the assay (between-run) varied between 11.3 and 15%. Application of the method in eleven breast tumors showed a great variation in the levels of telomerase enzymatic activity.


CONCLUSIONS
The proposed luminometric hybridization assay for the quantitative determination of telomerase enzymatic activity is highly sensitive and can be used for a large-scale prospective evaluation of clinical samples.
Kolioliou
M
M
Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, 15771, Athens, Greece.
Talieri
M
M
Lianidou
E S
ES
D001943:Breast Neoplasms / Q000201:enzymology; D002138:Calibration; D002624:Chemistry, Clinical / Q000379:methods*; D004274:DNA, Recombinant / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020014:K562 Cells; D008163:Luminescent Measurements; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D019098:Telomerase / Q000097:blood* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D004274:DNA, Recombinant; D019098:Telomerase