We just published a paper where we use the NanoBiT Technology to evaluate receptor activation, via β-arrestine 2 (βarr2) recruitment. More specifically, we applied the technology on the cannabinoid receptors. We used the developed cannabinoid reporter assay to evaluate the activity of synthetic cannabinoids and their metabolites. We also show a proof of concept for the application of the cannabinoid reporter assay as a first-line screening tool in urine.
Cannaert et al. Detection and activity profiling of synthetic cannabinoids and metabolites with a newly developed bio-assay. Anal Chem. 2016 Oct 25, PubMed PMID: 27779402. The publication is publically accessible at the following link: http://pubs.acs.org/doi/pdf/10.1021/acs.analchem.6b02600
This is what we did:
First, we cloned our proteins of interest, i.e. the cannabinoid receptors, CB1 and CB2, and βarr2 into the appropriate vectors, provided by Promega. Details on this can be found in Suppl. Data S1.
Second, we evaluated which combination is best suited for each receptor (giving the biggest increase in signal upon stimulation with an agonist). For the both cannabinoid receptors, this seemed to be different.
For CB1, this combination gave the biggest fold increase: CB1−LgBiT/SmBiT−βarr2
For CB2, this combination was selected: CB2−SmBiT/LgBiT−βarr2.
Subsequently, we show the concentration dependence of the cannabinoid reporter assay with a known agonist and antagonist. Next, we applied the cannabinoid reporter assay on a variety of compounds of which the cannabinoid activity was not known.
At last, we show a proof of concept that the cannabinoid reporter assay can be applied on urine samples of genuine users of synthetic cannabinoids.
If anyone has questions or wants more information, feel free to reply!!