(T1230-06-38) Performance of a Digoxigenin Antibody-Based Electrochemiluminescent Hybridization Assay: A HELISA Alternative for Low Femtomolar Detection of an Antisense Oligonucleotide

Purpose: With the rapid growth of antisense oligonucleotide (ASO) therapeutics, the demand for sensitive, specific, and reproducible oligonucleotide detection assays is increasing. Hybridization ELISA (HELISA) methods are popular choices for the development of pharmacokinetic (PK) assays for the detection of these ASO drugs. These assays typically use biotinylated capture probes bound to the surface of streptavidin-coated microplates and detection probes labeled with digoxigenin (DIG). Measurement of the analyte is then accomplished with an anti-DIG antibody labeled directly with a signaling molecule or with an enzyme that generates a signal during the processing of its substrate. Electrochemiluminescence (ECL)-based detection using SULFO-TAG labeled anti-DIG antibodies is an appealing alternative to increase assay sensitivity and improve reproducibility and has been shown to be one of the most effective methods for ASO detection by multiple groups. Here, we introduce three ECL hybridization assay protocols that further improve assay sensitivity compared to standard methods. These methods do not require extraction and use a newly optimized plate and buffer system. We also developed a system to increase sensitivity of these protocols.

Methods: Methods were developed for ECL-based detection of a 20-base-long DNA analyte using the formats of dual-probe hybridization, ligation with T4 DNA ligase, and a modified RNase protection assay. Sample pre-treatment, hybridization conditions, probe concentrations, and incubation times were optimized for each of the three assay formats. The specificities of the formats were assessed using 3’-shortened analyte metabolites, and matrix testing was performed in rabbit plasma samples. After assessing these formats, an augmented reporter system was established (N-PLEX ULTRA) to further improve assay sensitivity through signal amplification.

Results: After optimization of assay conditions, all three ECL hybridization assay methods showed excellent sensitivity with an estimated lower limit of detection (eLLOD) at or below 50 fM in both diluent and rabbit plasma. The ligation method proved to be the method of choice for 3’ discrimination, with only 0.1% off-target signal for the one base-shortened metabolite, whereas other test methods showed significantly higher off-target signals with the metabolites tested. Use of the N-PLEX ULTRA system enabled a further increase in sensitivity, with eLLODs approaching 1 fM.

Conclusion: ECL hybridization assay methods offer simple, highly sensitive, extraction-free, and high-throughput workflows for ASO PK assay development. The versatility of hybridization and enzymatic reaction-based approaches allow for the development of custom assays for the detection of many short oligonucleotide drugs, including DNA, RNA, or siRNA molecules. Further, multiple assay formats are adaptable to oligonucleotide drugs containing chemically modified bases. Lastly, the improvement in sensitivity with the N-PLEX ULTRA system sets a new standard for ultrasensitive oligonucleotide PK assay development.