A telomerase enzymatic assay that does not use polymerase chain reaction, radioactivity, or electrophoresis.
A telomerase assay has been developed for high-throughput screening in 96-well microtiter plates. A crude cell lysate which adds telomere repeats to a biotinylated DNA primer is the source of telomerase. The telomerase-extended primer is hybridized to a digoxigenin-labeled telomere antisense DNA probe. The hybrid is further processed by enzyme-linked immunosorbent assay (ELISA) as follows. The biotinylated hybrid is captured on streptavidin-coated microtiter plates. The immobilized hybrid is probed with alkaline phosphatase-antidigoxigenin and detected via chemiluminescent readout. The limit of detection of a chemically synthesized tetra-telomere repeat was about 10 attomoles. Apparent telomerase activity was detected in lysates of 293T cells. The signal to background for the assay (ratio of signal for the complete assay mixture divided by the signal for the assay mixture without primer) was around 10. An automated system that performed unattended runs of up to 17 96-well microtiter plates in 8h was constructed.
Kha
Hue
H
Amgen Inc., One Amgen Center, Mail Stop 29-1-A, Thousand Oaks, CA 91320-1799, USA.
Zhou
Wen
W
Chen
Kui
K
Karan-Tamir
Barbara
B
San Miguel
Tisha
T
Zeni
Lisa
L
Kearns
Karen
K
Mladenovic
Alex
A
Rasnow
Brian
B
Robinson
Murray
M
Wahl
Robert C
RC
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004586:Electrophoresis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction; D011868:Radioisotopes; D019098:Telomerase / Q000378:metabolism*
D017931:DNA Primers; D011868:Radioisotopes; D019098:Telomerase