Objectives: To develop a quantitative luminometric hybridization assay for the determination of telomerase activity in tissue and cell extracts.
Design and 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.