Development of a quantitative luminometric hybridization assay for the determination of telomerase activity

Objectives: To develop a quantitative luminometric hybridization assay for the determination of telomerase activity in tissue and eel extracts. Design and Methods: Quantification is based on the coamplification of telom eric repeats synthesized by telomerase along with a specifically designed r ecombinant DNA-internal standard (DNA-LS). The DNA-IS has a similar size an d the same primer recognition sites as the telomerase DNA products and diff ers from them only in a central 18 bp sequence. PCR products are captured o n microtiter wells via the biotin-streptavidin system and hybridized with t wo distinct digoxigenin-labeled oligonucleotide probes that are designed to recognize specifically telomerase products and DNA-IS. The hybrids are qua ntified by a luminometric reaction using an antidigoxigenin antibody conjug ated to alkaline phosphatase. The hybridization assay was validated with th e MCF-7 breast carcinoma and leukemia K-562 cell lines and a synthetic telo merase product (R-8), Results: Luminescence ratios for telomerase products and DNA-IS were linear ly related to the concentration of the pre-PCR product synthesized by telom erase (R-8), in the range of 0.0005 to 10 pM. The overall reproducibility o f the assay (between-run) varied between 11.3 and 15%. Application of the m ethod in eleven breast tumors showed a great variation in the levels of tel omerase enzymatic activity. Conclusions: The proposed luminometric hybridization assay for the quantita tive determination of telomerase enzymatic activity is highly sensitive and can be used for a large-scale prospective evaluation of clinical samples. (C) 2001 The Canadian Society of Clinical Chemists. All rights reserved.