This article is based on a poster originally authored by Bossé R., Deschamps F., Fafard C., Lévesque C., and Roby P.
Abstract
ENLIGHT OMEGA™ (Oxygen MEdiated/Gated Assay) is a non-wash (homogeneous) assay technology that allows users to perform a wide variety of biochemical and cell-based assays. ENLIGHT OMEGA relies on a unique combination of Donor (Sensibeads) and Acceptor (Chemibeads) nanoparticles, each embedded with novel proprietary chemicals and layered with improved hydrogel coatings, allowing for optimal bioconjugations.
OMEGA relies on principles similar to those of LOCI™ previously described by Ullman et al.*. This communication describes an efficient strategy to detect and characterize compounds that could interfere with ENLIGHT OMEGA. We describe how TruActiv ID™ 545 and 615 kit components can be used in combination to detect interfering compounds such as singlet oxygen (102) quenchers, biotin mimetics, compound aggregates, and PAINS in a single well.
*Proc Natl Acad Sci US A. 1994 Jun 7;91(12):5426-30
1. Principles of OMEGA
Image Credit: ELRIG (UK) Ltd.
ENLIGHT OMEGA assays allow users to measure the interaction occurring between two binding partners (A and B) respectively captured onto Sensibeads 680 and Chemibeads 615 nanoparticles. Singlet oxygen, produced upon excitation of the Sensibeads at 680 nm, travels up to 200 nm in aqueous solution and reacts with specific compounds embedded in the Chemibeads when located in close proximity. The latter beads then emit a signal detectable at 615 nm, which is proportional to the extent of interaction prevailing between binding partners A and B. Reading at a wavelength below that is used for excitation allows for the detection of high-quality signals (e.g., S:B values up to 1000:1).
2. Compound Interference
Image Credit: ELRIG (UK) Ltd.
Any detection technology is susceptible to compound interference. The main classes of compounds known to interfere with OMEGA are 1) biotin mimetics, 2) 102 quenchers such as NaN3, and some transition metals (Cu2+, Zn2+, Fe2+). The presence of these compounds produces an OMEGA signal decrease proportional to their concentrations.
3. OMEGA Duplexing
Image Credit: ELRIG (UK) Ltd.
Multiplexing using Chemibeads 545 (Tb3+) and 615 (Eu3+). Due to their distinct emissions and limited overlap, Chemibeads 545 and 615 can be used simultaneously in the same well to detect two concurring events.
4. TruActiv ID™ Duplexing Concept
Image Credit: ELRIG (UK) Ltd.
TruActiv ID kits are composed of biotinylated-Chemibeads (615 or 545) and Streptavidin Sensibeads 680. Adding compounds to a pre-bound bead complex allows one to detect 102 quenchers and compound aggregates.
In addition to these interfering molecules, it is possible to detect biotin mimetics and PAINS (pan assay interference compounds) by incubating compounds with Streptavidin Sensibeads first, followed by the addition of biotinylated Chemibeads. Using a combination of biotinylated Chemibeads 615 and 545 together with Streptavidin Sensibeads allows one to detect and differentiate interfering compounds listed above in a single well!
5. Biotin Mimetic Test
Image Credit: ELRIG (UK) Ltd.
Protocol
Biotin-Chemibeads 545 (2 ug/mL) and Streptavidin Sensibeads 680 (10 μg/mL) are premixed for 30 minutes. The following reagents are then added in order in a 384-well white opaque microplate:
- 10 μL of the premixed beads
- 5 μL biotin (0-10 μM) (incubate 30 minutes @ RT)
- 10 μL of biotin-Chemibeads 615 (8 μg/mL) (incubate 30 minutes @ RT)
Sequential readings were then performed on multilabel readers at 610-635 nm and 535-560 nm.
Results
Increasing concentrations of biotin produced a proportional signal decrease (IC50 10 nM) measurable at 615 nm when biotinylated Chemibeads 615 were added last. The signal generated at 545 nm by the biotin- Chemibeads 545 pre-bound to the Streptavidin 680 nm was not affected by the presence of biotin.
6. 102 QuenchTest
Image Credit: ELRIG (UK) Ltd.
Protocol
Biotin-Chemibeads 545 (2 ug/mL) and Streptavidin Sensibeads 680 (10 μg/mL) were then premixed for 30 minutes. The following reagents were then added in order in a 384 well white opaque microplate:
- 10 μL of the premixed beads
- 5 μL biotin (0-10 μM) (incubate 30 minutes @ RT)
- 10 μL of biotin-Chemibeads 615 (8 μg/mL) (incubate 30 minutes @ RT)
Sequential readings were then performed on multilabel readers at 610-635 nm and 535-560 nm.
Results
Increasing concentrations of sodium azide (NaN3) produced a proportional signal decrease (IC50 = 10 μM) measurable at 615nm when biotinylated Chemibeads 615 were added last. An identical trend was observed at 545 nm with biotin-Chemibeads 545 pre-bound to Streptavidin 680 nm.
7. TruActiv ID Duplexing Validation
Image Credit: ELRIG (UK) Ltd.
Protocol
Biotin-Chemibeads 545 (2 ug/mL) and Streptavidin Sensibeads 680 (10 μg/mL) were premixed for 30 minutes. The following reagents were added in order in a 384 well white opaque microplate:
- 10 μL of the premixed beads
- 5 μL biotin or NaN3, (10 μM) or buffer (incubate 30 minutes @ RT)
- 10 μL of biotin-Chemibeads 615 (8 μg/mL) (incubate 30 minutes @ RT)
Sequential readings were performed on a multilabel reader at 610-635 nm and 535-560 nm.
384 Well Plate Test
Biotin and NaN3, were both used at 10 μM and distributed uniformly in specific wells covering a wide area within a 384 well microplate. A 10 μM concentration represents approximately the IC95 of biotin and the IC50 of NaN3, as previously observed with the biotin-mimetic and 102 quench tests.
8.TruActiv ID Duplexing Validation
Image Credit: ELRIG (UK) Ltd.
Results
The wells of a 384 white opaque microplate were filled with TruActiv ID 545 and 615 kits components, control compounds (biotin, NaN3) or assay buffer (negative control) as described in panel 7. All 12 wells spiked with NaN3 were identified in both 545 nm and 615 nm channels for a 100 % accuracy, while the 10 wells spiked with biotin were only identified in the 615 nm channel for a 100 % accuracy.
9. Summary
We developed a simple and efficient method to rapidly identify potential compounds interfering with ENLIGHT OMEGA :
- Components of TruActiv ID 545 and 615 kits were used: biotin- Chemibeads 545, biotin-Chemibeads 615 and Streptavidin Sensibeads 680.
- Adding compounds to premixed biotin-Chemibeads 545 and Streptavidin Sensibeads 680 allows for the identification of 1O2 quenchers.
- Adding compounds to premixed biotin-Chemibeads 545 and Streptavidin Sensibeads 680, followed by the addition of biotin-Chemibeads 615, allowing for the selective identification of biotin mimetics.
- Both approaches described above can be duplexed in a single well.
This method could be proactively used to clean up an entire screening compound library and maximize the quality of an HTS campaign performed with ENLIGHT OMEGA.
About ELRIG (UK) Ltd.
The European Laboratory Research & Innovation Group (ELRIG) is a leading European not-for-profit organization that exists to provide outstanding scientific content to the life science community. The foundation of the organization is based on the use and application of automation, robotics, and instrumentation in life science laboratories, but over time, we have evolved to respond to the needs of biopharma by developing scientific programs that focus on cutting-edge research areas that have the potential to revolutionize drug discovery.
Comprised of a global community of over 12,000 life science professionals, participating in our events, whether it be at one of our scientific conferences or one of our networking meetings, will enable any of our community to exchange information, within disciplines and across academic and biopharmaceutical organizations, on an open access basis, as all our events are free-of-charge to attend!
Our Values
Our values are to always ensure the highest quality of content and that content will be made readily accessible to all, and that we will always be an inclusive organization, serving a diverse scientific network. In addition, ELRIG will always be a volunteer led organization, run by and for the life sciences community, on a not-for-profit basis.
Our Purpose
ELRIG is a company whose purpose is to bring the life science and drug discovery communities together to learn, share, connect, innovate and collaborate, on an open access basis. We achieve this through the provision of world class conferences, networking events, webinars and digital content.
Sponsored Content Policy: AZoLifeSciences publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of AZoLifeSciences which is to educate and inform site visitors interested in life science news and information.