Development of an Ultrasensitive Noncompetitive Hybridization–Ligation Enzyme-Linked Immunosorbent Assay for the Determination of Phosphorothioate Oligodeoxynucleotide in Plasma

doi:10.1006/abio.2002.5576

Analytical Biochemistry

Volume 304, Issue 1, 1 May 2002, Pages 19-25

https://doi.org/10.1006/abio.2002.5576 Get rights and content

Abstract

An ultrasensitive noncompetitive hybridization–ligation heterogeneous enzyme-linked immunosorbent assay was developed for the quantitation of antisense phosphorothioate oligodeoxynucleotides in plasma using a 96-well plate format. The principle of the assay is based on heterogeneous noncompetitive binding of the analyte to a template probe, followed by addition of signal probe via ligation and detection using a fluorescence microtiter plate reader. The result showed no significant interference noted from untreated human plasma. In addition, the method is selective for the specific sequence tested (ISIS 2302) and cross-reactivity toward the 3′-metabolites is minimal (<0.22%). A linear range of 0.05 to 2 nM (r > 0.99) was obtained in human plasma for ISIS 2302. Intraday and interday accuracy for the method was found to be within 80–120% of actual value. Intraday and interday precision has a percentage coefficient of variation less than 20%. The lower limit of quantitation of the method was 0.05 nM (0.05 pmol/ml) with 100 μL plasma or an absolute amount of 5 fmol. In summary, the assay was demonstrated to be specific, accurate, precise, and sensitive for the quantitation of ISIS 2302 in human plasma and was applied to the analysis of plasma samples in pharmacokinetic studies.

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¹: To whom correspondence should be addressed at Isis Pharmaceuticals, 2292 Faraday Avenue, Carlsbad, CA 92008. Fax: +1 (760) 603-3862. E-mail: [email protected].

²: Present address: Bayer Corporation, Dept. of Biotechnology, 800 Dwight Way, Box 1986, Berkeley, CA 94701.

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Regular ArticleDevelopment of an Ultrasensitive Noncompetitive Hybridization–Ligation Enzyme-Linked Immunosorbent Assay for the Determination of Phosphorothioate Oligodeoxynucleotide in Plasma

Abstract

J. Biol. Chem.

Gastroenerology

J. Chromatogr. Biomed. Appl.

J. Chromatogr.

Anal. Biochem.

J. Chromatogr. A

Anal. Biochem.

Biochem. Biophys. Res. Commun.

Biochem. Pharmacol.

In vitro and in vivo activity of antisense inhibitors of ras: potential for clinical development

Anticancer Drug Des.

Regular Article
Development of an Ultrasensitive Noncompetitive Hybridization–Ligation Enzyme-Linked Immunosorbent Assay for the Determination of Phosphorothioate Oligodeoxynucleotide in Plasma