A Specific Picomolar Hybridization-Based ELISA Assay for the Determination of Phosphorothioate Oligonucleotides in Plasma and Cellular Matrices View Full Text


Ontology type: schema:ScholarlyArticle     


Article Info

DATE

2006-05-25

AUTHORS

Xiaohui Wei, Guowei Dai, Guido Marcucci, Zhongfa Liu, Dale Hoyt, William Blum, Kenneth K. Chan

ABSTRACT

PurposeTo develop and validate an ultrasensitive and specific hybridization-based enzyme-linked immunosorbent assay method for quantification of two phosphorothioate oligonucleotides (PS ODNs) (G3139 and GTI-2040) in biological fluids.MethodsThis assay was based on hybridization of analytes to the biotin-labeled capture ODNs followed by ligation with digoxigenin-labeled detection ODN. The bound duplex was then detected by anti-digoxigenin-alkaline phosphatase using Attophos® (Promega, Madison, WI, USA) as substrate. S1 nuclease and major factors such as the hybridization temperature, concentration of capture probe, and the use of detergent were evaluated toward assay sensitivity, selectivity, and accuracy.ResultsThe method is selective to the parent drugs with minimal cross-reactivity (<6%) with 3′-end deletion oligomers for both G3139 and GTI-2040. A linear range of 0.05 to 10 nM (r2 > 0.99) was observed for GTI-2040 in a variety of biological matrices. For both G3139 and GTI-2040, the within-day precision and accuracy values were found to be <20% and 90–110%, respectively; the between-day precision and accuracy were determined to be <20% and 90–120%. Addition of S1 nuclease combined with washing step greatly improved the assay linearity and selectivity. The utility of this assay was demonstrated by simultaneous determination of GTI-2040 in plasma and its intracellular levels in treated acute myeloid leukemia patients.ConclusionsThe validated hybridization enzyme-linked immunosorbent assay method is specific for quantitation of PS ODNs in biological samples to picomolar level. This method provides a powerful technique to evaluate plasma pharmacokinetics and intracellular uptake of PS ODNs in patients and shows its utility in clinical evaluations. More... »

PAGES

1251-1264

Identifiers

URI

http://scigraph.springernature.com/pub.10.1007/s11095-006-0082-3

DOI

http://dx.doi.org/10.1007/s11095-006-0082-3

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1050107597

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/16718617


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