Application

The TeloTAGGG Telomerase PCR ELISA^PLUS has been used to detect telomerase activity in urine, frozen sections of head and neck squamous cell carcinomas and tumor margin tissues.

Features and Benefits

Ready-to-use master mixes: the kit contains all of the essential compounds as master mixes Reliability: Positive control contained in the kit Specificity: The use of hybridization probes ensures specific detection of the applied telomerase reaction product Quantitative results: The assay concept allows semi-quantitative determination of telomerase levels

General description

The TeloTAGGG Telomerase PCR ELISA^PLUS kit provides a way to perform a highly sensitive photometric enzyme immunoassay to detect telomerase activity, using nonradioactive techniques. The TeloTAGGG Telomerase PCR ELISA^PLUS is designed for use in life science research studies in cell extracts from cell cultures and other biological research samples. The TeloTAGGG Telomerase PCR ELISA^PLUS utilizes single-tube reaction mixtures, similar to the TeloTAGGG Telomerase PCR ELISA, but also contains an optimized internal standard and control template, which allows the TeloTAGGG Telomerase PCR ELISA^PLUS to quantitatively measure the amount of telomerase reaction product. It uses the Telomeric Repeat Amplification Protocol (TRAP).

Legal Information

TeloTAGGG is a trademark of Roche

Manufactured under license from Geron Corporation.

Other Notes

For life science research only. Not for use in diagnostic procedures.

Packaging

1 kit containing 16 components.

Preparation Note

Working solution: Solution 10: Washing Buffer, 1xDilute an appropriate volume of Washing buffer, 10x concentrated (bottle 10) with autoclaved double-distilled water (1:10) and mix thoroughly. Approximately 5 ml of the diluted Washing buffer are needed for one reaction.Stable at 2 to 8 °C for 1 month.Solution 11: Anti-DIG HRP, bottle 11 stock solutionReconstitute the lyophilizate in 240 µl autoclaved double-distilled water. This results in an antibody conjugate concentration of 0.5 U/ml.Stable at 2 to 8 °C for 6 months. Do not freeze! Do not add sodium azide!Solution 11: Anti-DIG HRP, working solutionTo prepare the working solution, dilute an appropriate amount of the reconstituted anti-DIG-HRP (solution 11) with Conjugate dilution buffer (solution 12) to a final concentration of 10 mU/ml (e.g., 200 µl antibody solution and 9.8 ml of Conjugate dilution buffer).Prepare immediately before use. Do not store.Storage conditions (working solution): Washing buffer, 1x:Stable at 2 to 8 °C for 1 month.Anti-DIG HRP, stock solution:Stable at 2 to 8 °C for 6 months. Do not freeze. Do not add sodium azide.Anti-DIG HRP, working solution:Prepare immediately before use. Do not store.

Principle

The test principle can be divided into the following steps:Step 1: Elongation/amplificationIn the first step, telomerase adds telomeric repeats (TTAGGG) to the 3′-end of the biotin-labeled synthetic P1-TS primer. These elongation products, as well as the Internal standard (IS) included in the same reaction vessel, are amplified by PCR using the primers P1-TS and the anchor-primer P2. PCR products derived from telomerase-mediated elongation products in the first step contain the telomerase-specific six nucleotide increments, while the Internal standard (IS) generates a 216-bp PCR product.Step 2: Detection by ELISAThe PCR products are split into two aliquots, denatured, and hybridized separately to digoxigenin-(DIG)-labeled detection probes that are specific for the telomeric repeats (P3-T) and for the Internal standard (IS) (P3-Std), respectively. The resulting products are immobilized via the biotin label to a streptavidin-coated microplate. Immobilized amplicons are then detected with an antibody against digoxigenin, which is conjugated to horseradish peroxidase (Anti-DIG-HRP) and the sensitive peroxidase substrate TMB.

Specifications

Assay time: Approximately 7 hoursMeasuring range: The linear measuring range of the kit is from 10 - 2,000 cells (in a model system using 293 cells)Sample material: Cell cultures and biological research samplesSensitivity: Detects ﹤10 cell equivalents (in a model system, using 293 cells)