Jr. Deverre et al., A COMPETITIVE ENZYME HYBRIDIZATION ASSAY FOR PLASMA DETERMINATION OF PHOSPHODIESTER AND PHOSPHOROTHIOATE ANTISENSE OLIGONUCLEOTIDES, Nucleic acids research, 25(18), 1997, pp. 3584-3589
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 an
alogs. Assays were performed in 96-well microtiter plates, The phospho
diester sense sequence was covalently bound to the microwells. The 5'-
biotinylated antisense sequence was used as tracer, The principle of t
he assay involves competitive hybridization of tracer and antisense nu
cleotide to the solid phase-immobilized sense oligonucleotide. Solid p
hase-bound tracer oligonucleotide was assayed after reaction with a st
reptavidin-acetylcholinesterase conjugate, using the colorimetric meth
od of Ellman, As in competitive enzyme immunoassays, coloration was in
versely related to the amount of analyte initially present in the samp
le. The limit of quantification was 900 pM for phosphodiester antisens
e oligonucleotide using a 100 mu l 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 f
or in vitro screening of oligonucleotide hybridization potency in biol
ogical fluids and for measuring the plasma pharmacokinetics of phospho
rothioate and phosphodiester sequences.