A novel magnetic bead‐based assay with high sensitivity and selectivity for analysis of telomerase in exfoliated cells from patients with bladder and colon cancer

Abstract

Telomerase activity is elevated in more than 85% of cancer cells and absent in most of the normal cells and thus represents a potential cancer biomarker. We report its measurement in colon and bladder cancer cells captured using antibody-coated magnetic beads. The cells are lysed and telomerase activity is detected using a biosensor assay that employs an oligonucleotide containing the telomerase recognition sequence also covalently coupled to magnetic beads. Telomerase activity is measured by the incorporation of multiple biotinylated nucleotides at the 3'-end of the oligonucleotide strands during elongation which are then reacted with streptavidin-conjugated horseradish peroxidase. A luminescent signal is generated when hydrogen peroxidase is added in the presence of luminol and a signal enhancer. LOD experiments confirm sensitivity down to ten cancer cell equivalents. The telomerase assay reliably identified patient samples considered by an independent pathological review to contain cancer cells. Samples from normal healthy volunteers were all telomerase negative. The assay, which is amenable to automation, demonstrated high sensitivity and specificity in a small clinical cohort, making it of potential benefit as a first line assay for detection and monitoring of colon and bladder cancer.

Analysis of telomerase activity based on a spired DNA tetrahedron TS primer

“…With the presence of telomerase, the TS primer was elongated using mixed dNTPs including biotin labelled dATP (Weizmann et al, 2004;Rothacker et al, 2007). The subsequently added avidin-HRP would be captured by the biotin, and then catalyzed the electrochemical reduction of TMB to consequently generate an amplified current signal.…”

Protein Analytical Assays for Diagnosing, Monitoring, and Choosing Treatment for Cancer Patients

“…While rarely expressed in most normal cells, telomerase is overactive in more than 85% of tumors, making it a possible general cancer biomarker [126]. Telomerase activity is most commonly measured by the telomere repeat amplification protocol (TRAP).…”

“…The TRAP assay requires radioisotopes and relies on PCR, which can be contaminated by telomerase inhibitors, heme components from blood, or other molecules inhibiting the reaction and causing false negatives [126]. It is also time consuming and can generate false positives due to primer dimers [129].…”

“…Exposure to telomerase containing cell lysate in the presence of biotin labeled dUTP resulted in extension of these sequences. Streptavidin-HRP labeling of biotin then detected the biotinylated sequences [126]. Another magnetic bead assay eliminated the need for PCR amplification by increasing sensitivity with an ECL probe containing 100 ECL signaling molecules per telomerase product.…”

Feasibility studies for assaying alpha-fetoprotein using antibody-activated magnetic nanoparticles

“…[1][2][3] Some other groups have explored advanced technologies. [4][5][6][7] Several years ago, some authors proposed an assay technology called immunomagnetic reduction (IMR). 5 In IMR, the reagent is a solution of homogeneously dispersed magnetic nanoparticles, which are coated with hydrophilic surfactants (eg, dextran) and bioprobes (eg, antibodies).…”