Quantifying Oligos in Clinical Samples
Hybridization assays for quantifying oligo drug levels in biological samples have advantages over conventional chromatographic techniques such as capillary gel electrophoresis or HPLC. The main advantage is the high sensitivity of ELISA assays, the small sample volumes required, and the ease of use, with little or no sample clean-up required prior to assay processing.
In fact, “oligo levels can be measured in homogenized tissue samples without any prior extraction,” says Helen Legakis, research scientist in immunochemistry laboratory services at Charles River Laboratories. In contrast, tissue samples are subjected to sonication and enzyme digestion to break down the tissue membranes.
As oligo-based therapeutics are mainly administered parenterally, a high assay sensitivity is essential to characterize their pharmacokinetic and toxicokinetic properties following systemic administration. This is particularly important at “the terminal elimination phase, characterized after near-complete distribution of the drug has occurred,” notes Legakis.
To achieve optimal sensitivity, Charles River designs its probes to include Locked Nucleic Acid inserts, which contain a rigid bicyclic structure in the ribofuranose ring. This reduces the background noise of the assay and increases the thermal melting temperature of the oligo analyte and the complementary probe. The increased binding affinity to the probe prevents competitive re-annealing of double-stranded analytes to their complementary strand, Legakis explains.
Looking to the future, Legakis sees a growing demand for multiplexing technologies coupled to an ELISA-based assay format. With the emergence of multiple therapeutic targets, these methods will be able to measure more than one analyte in a multianalyte drug cocktail in a single well.