(T1130-01-01) Strategies for Measuring Oligonucleotides Using a Fully Automated Microfluidic Immunoassay System

Purpose: The rapidly growing field of oligonucleotide medicine provides unique opportunities to treat genetic diseases and provide new treatment and vaccines for viral diseases. As of 2021, there are 15 FDA approved oligonucleotide-based drugs (excluding vaccines), and many more are currently in clinical trials. These drugs are often structural variants of DNA or RNA which increases resilience, uptake and target affinity. Pharmacokinetic (PK) and pharmacodynamic (PD) measurements of these drugs in clinical trials can be performed by utilizing the base pairing capabilities in hybridization assays which mimic an ELISA format. However, these methods are often time and labor intensive. Hence, transferring such methods to an automated microfluidics system could potentially increase throughput and decrease runtime. In this study, two assay formats were evaluated to detect an analyte mimicking the anti-sense oligonucleotide drug casimersen (Amondys 45™) using a microfluidic CD-based immunoassay platform and a labeled oligonucleotide probe.

Methods: Two assay approaches were evaluated both in a manual then automated format, the dual hybridization assay (DHA) and the nuclease-dependent cutting assay (NCA). The DHA utilizes one capture oligo and one detection oligo. If not held in place by the analyte, the detection oligo is washed away. The NCA utilizes a full-length oligo probe biotinylated in one end and with a conjugated fluorophore on the other end. Single stranded probes that are unbound or partially hybridized are cleaved by nuclease and the detection molecule is washed away.

Results: The manual DHA was successfully performed with optimization of incubation times, reagent concentrations and buffers. The assay was easily transferred to the automated microfluidic system. The assay performance for detection of the oligo analyte in 50% human serum in 6 runs was 2 pM LLOQ in neat serum, 4.5% CV within runs, 1.9% CV between runs, 4.9% total CV. The manual NCA was successfully performed manually however automation revealed difficulties with large variations between replicates that was not resolved by adjusting salt concentrations, addition of BSA or serum, and the precision remained too low for usability.

Conclusion: The DHA assay for detection of the oligonucleotide therapeutic casimersen works well on the automated immunoassay system with an LLOQ of 2 pM in neat serum. Assay runtime is about 2.5 hours of which the vast majority is automated, representing a significant savings of hands-on and total assay time.

References: Thakur S, Sinhari A, Jain P and Jadhav HR (2022) A perspective on oligonucleotide therapy: Approaches to patient customization. Front. Pharmacol. 13:1006304. doi: 10.3389/fphar.2022.1006304


ELISA and hybridization immunoassay formats for oligo detection.


Assay performance for manual and automated hybridization immunoassay format.


Assay performance for manual and automated nuclease cutting assay format.