Highly-Sensitive Hybridization ELISA Development for Oligonucleotide Quantitation at Picogram Levels

Oligonucleotide modifications and delivery advances have increased drug half-life, necessitating more sensitive pharmacokinetic methods. Electrochemiliuminescent methods provide the most sensitive platform for the quantitation of oligonucleotides but are sole sourced. ELISA provides an alternative to ECL but has lower sensitivity. We developed and qualified an ELISA for oligonucleotide quantitation with sensitivity of 12.5 picogram/milliliter, which is ~10-40 fold lower compared to typical ELISA sensitivity for oligonucleotide methods. Increased sensitivity was achieved through advanced probe design and optimization to increase binding affinity. However, lower amounts of probe utilized to minimize background and increase signal to noise ratio can cause significant prozone effects due to the high levels of parent compound. Prozone effects were mitigated with probe concentration optimization relative to anticipated matrix Cmax while maintaining assay sensitivity. Thus, ELISA can provide sensitivity for oligonucleotide quantitation that is comparable with ECL methods and provides an alternative for PK methods supporting oligonucleotide trials.