Nucleic Acids Research, Vol 25, Issue 18 3584-3589, Copyright © 1997 by Oxford University Press
JR Deverre, V Boutet, D Boquet, E Ezan, J Grassi and JM Grognet
An enzyme competitive hybridization assay was developed and validated for
determination of mouse plasma concentrations of a 15mer antisense
phosphodiester oligodeoxyribonucleotide and of two phosphorothioate
analogs. Assays were performed in 96-well microtiter plates. The
phosphodiester sense sequence was covalently bound to the microwells. The
5'-biotinylated antisense sequence was used as tracer. The principle of the
assay involves competitive hybridization of tracer and antisense nucleotide
to the solid phase-immobilized sense oligonucleotide. Solid phase- bound
tracer oligonucleotide was assayed after reaction with a
streptavidin-acetylcholinesterase conjugate, using the colorimetric method
of Ellman. As in competitive enzyme immunoassays, coloration was inversely
related to the amount of analyte initially present in the sample. The limit
of quantification was 900 pM for phosphodiester antisense oligonucleotide
using a 100 microl volume of plasma without extraction. Cross-reactivity
was negligible after a four base deletion in either the 3'or 5'position.
The assay was simple and sensitive, suitable for in vitro screening of
oligonucleotide hybridization potency in biological fluids and for
measuring the plasma pharmacokinetics of phosphorothioate and
phosphodiester sequences.
ARTICLES
A competitive enzyme hybridization assay for plasma determination of phosphodiester and phosphorothioate antisense oligonucleotides
Service de Pharmacologie et d'Immunologie, DRM/DSV, CEA-Saclay, F-91191 Gif-sur-Yvette, France. deverre@dsvidf.cea.fr
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