A specific picomolar hybridization-based ELISA assay for the determination of phosphorothioate oligonucleotides in plasma and cellular matrices.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Wei
Xiaohui
X
Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, USA.
Dai
Guowei
G
Marcucci
Guido
G
Liu
Zhongfa
Z
Hoyt
Dale
D
Blum
William
W
Chan
Kenneth K
KK
D000208:Acute Disease; D000818:Animals; D015342:DNA Probes; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D007951:Leukemia, Myeloid / Q000097:blood / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D051379:Mice; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense / Q000032:analysis* / Q000097:blood / Q000493:pharmacokinetics; D051381:Rats; D015203:Reproducibility of Results; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013696:Temperature; D013873:Thionucleotides / Q000032:analysis* / Q000097:blood / Q000652:urine
D015342:DNA Probes; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D013873:Thionucleotides; C464617:GTI2040; C408162:oblimersen; D015719:Single-Strand Specific DNA and RNA Endonucleases