Detection of human immunodeficiency virus type 1 reverse transcriptase using aptamers as probes in affinity capillary electrophoresis.
An affinity capillary electrophoresis/laser-induced fluorescence (CE/LIF) assay was developed for direct and specific detection of reverse transcriptase (RI) of the type 1 human immunodeficiency virus (HIV-1) using fluorescently labeled single-stranded DNA aptamers as probes. The aptamer used (RT 26) is specific for HIV-1 RT, and it exhibited no cross-reactivity with RTs of the enhanced avian myeloblastosis virus (AMV), the Moloney murine leukemia virus (MMLV), or denatured HIV-1 RT. An affinity complex of RT 26-HIV-1 RT was readily formed, and calibration curves were linear up to 50 nM (6 microg/mL) HIV-1 RT concentration, with both the free probe and complex peak usable for analytical quantitation. Cell culture media (RPMI with 10% fetal bovine serum) interfered with the assay and aptamer-HIV-1 RT binding. Nonspecific binding was observed in low or undiluted culture, necessitating at least 100-fold dilution for analysis of raw culture samples.
Pavski
V
V
Environmental Health Sciences Program, Department of Public Health Sciences, University of Alberta, Edmonton, Canada.
Le
X C
XC
D002138:Calibration; D004277:DNA, Single-Stranded; D019075:Electrophoresis, Capillary; D005456:Fluorescent Dyes; D054303:HIV Reverse Transcriptase / Q000032:analysis*; D007202:Indicators and Reagents; D013050:Spectrometry, Fluorescence
D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D054303:HIV Reverse Transcriptase