Solution
One of our sponsors needed to achieve a reproducible PK assay with high sensitivity for their oligonucleotide therapeutic. Here, we performed a Meso Scale Discovery based assay combined with oligonucleotide hybridization.
Our veteran team optimized every step of this oligonucleotide hybridization ECL method to improve the assay performance and robustness.
To begin with, our team utilized a checkerboard approach to determine the optimal concentrations for coating and detection oligonucleotides, blocking conditions, temperature for oligonucleotide denaturation, annealing and hybridization, and duration of incubation. We also worked out appropriate sample dilution as this analyte posed a hook effect at higher concentrations.
MSD multi-array plate was coated overnight with the capture oligonucleotide. Standards and samples were prepared, heat-inactivated, and combined with biotinylated detection oligonucleotide. Next, this mixture was annealed on a thermocycler and incubated in the coated plate to allow oligonucleotide hybridization. Afterward, we washed off the unbound oligonucleotides and detected the ECL signal from hybridized oligonucleotide with streptavidin-SULFO-TAG on an MSD imager.