A Specific Picomolar Hybridization-Based ELISA Assay for the Determination of Phosphorothioate Oligonucleotides in Plasma and Cellular Matrices
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To 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.
This 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.
The 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.
The 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.
Key Wordshybridization–ligation ELISA intracellular drug level pharmacokinetics phosphorothioate oligonucleotides
acute myeloid leukemia
- AS ODN
area under the curve
total body clearance
steady state concentration
coefficient of variation
enzyme-linked immunosorbent assay
lower limit of quantification
limit of detection
messenger ribonucleic acid
peripheral blood mononuclear cell
- PS ODN
red blood cells
- RNase H
We acknowledge the support by NIH R21CA105879 and UO1 CA 76576.
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