Our experience with the NanoBiT

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  • Last Post 22 August 2017
Annelies.Cannaert posted this 10 November 2016

Dear all,

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!!

 

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alandrem posted this 22 August 2017

Very nice application of this NanoBiT assay recently published. Congratulations!

Cannaert et al. (2017) Activity-based detection of consumption of synthetic cannabinoids in authentic urine samples using a stable cannabinoid reporter system. Analytical Chemistry DOI: 10.1021/acs.analchem.7b02552. http://pubs.acs.org/doi/abs/10.1021/acs.analchem.7b02552

 

Annelies.Cannaert posted this 01 December 2016

At the moment we started with the development of the NanoBiT assay for our system, we did not have any positive/negative controls.

We used our receptor-tagged with SmBiT and LgBiT (single transfected) as a negative controle. When expressed alone we did not see an increase in signal upon stimulation with an agonist (see figure 2 in the publication: no difference between blank and 1µM JWH-018).

What we did observe is that the basal signal for the single transfected LgBiT-tagged receptor is higher than single transfected the SmBiT-tagged receptor (maybe not so clear on the figure 2). This is not suprising. That autoluminescence might be due to the low level of catalytic activity associated with LgBiT.

When both the LgBiT and SmBiT were present (double transfection with receptor and b-arrestin-fusion proteins), our basal signal was increased (you can see that in figure 2: the white bars -blank- for the 4 combinations are higher). We assume this increased signal is due to the spontaneous auto-complementation between LgBiT and SmBiT. This higher basal signal did not pose a problem as when stimulated with our agonist, we can see a clear increase in signal. As we did see such an good concentration dependent interaction, we assumed our system worked (kind of a positive control).

All fusion proteins are expressed via the HSV-TK promotor (the promotor that was in the NanoBiT vectors that were provided by Promega).

BBinkowski at Promega posted this 30 November 2016

"Did you use the negative and/or positive controls for your NanoBiT assays? In your paper, you only mention the blank as a negative control and normalization reference. I wonder how was your background signal. We are working on similar stuff and experience some problems with the basal luminescence from a LgBiT-tagged GPCR or b-arrestin."

Mokros: Are you expressing via the HSV-TK promoter or a stronger promoter? Do you see a high basal signal from the LgBiT fusion expressed alone or only when you co-express with the SmBiT fusion? Do you see a signal increase after agonist treatment?

mokros posted this 30 November 2016

Did you use the negative and/or positive controls for your NanoBiT assays? In your paper, you only mention the blank as a negative control and normalization reference. I wonder how was your background signal. We are working on similar stuff and experience some problems with the basal luminescence from a LgBiT-tagged GPCR or b-arrestin.

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