Science is the practice of figuring out how things work and then using that knowledge to further our understanding or to create tools that can solve problems facing the world. Bioluminescent tools and assays are examples of science doing all these things. Bioluminescence is the light-yielding (luminescence) chemical reaction that is used by many lifeforms. When fireflies flicker in the twilight, they are using bioluminescence to flash on and off. Chemically, bioluminescence happens when an enzyme called luciferase acts on a light-emitting compound, luciferin, in the presence of adenosine triphosphate (ATP), magnesium and oxygen.
For scientists, bioluminescence can serve as a tool to help them understand many cellular functions. Since few animal or plant cells produce their own light, there is little to no background signal (light) to be concerned about. This lack of background means that all light coming from the sample can be measured. In fact, bioluminescence is often a preferred tool for scientists because it does not require an external light source or special filters, which are required for fluorescence-based technologies.
Promega scientists have developed bioluminescent tools and assays to support leading edge scientific research for decades, beginning in 1990 with the Luciferase biosensor technology based on firefly luciferase. Luciferase is a wonderful tool for studying how enzymes work because its output (light) is so easy to measure: samples are placed into a special instrument called a luminometer, and the amount of light being produced (Relative Light Units) is recorded. Bioluminescence technology can be configured to measure a variety of cellular biology, ranging from cell health to enzyme activity down to the specific event of turning a gene on or off. The advent of new techniques for genetic manipulation, along with an enhanced understanding of bioluminescence and the discovery and engineering of better luciferases, enables science to use bioluminescence in even more unique ways.
More recent developments from Promega include the small NanoLuc® luciferase, developed using directed evolution of the luciferase from the deep-sea shrimp Oplophorus gracilirostris (1). The enzyme is 100X brighter than traditional luciferase, which enables more sensitive detection of intracellular events than other bioluminescent reporters, and its small size means it can “fit” in situations where larger reporter molecules may not be as useful. These properties allow NanoLuc® luciferase to be used to detect intracellular events in ways not possible before (2–4). For example, researchers at the University of Wisconsin-Madison used NanoLuc® luciferase to create a luminescent influenza virus, enabling them to visualize the progression of influenza infection in live mice by looking for the light-emitting cells that contain the virus (5).
In 2015, Promega introduced a new tool based on NanoLuc® Luciferase: NanoLuc® Binary Interaction Technology (NanoBiT®; 2). The technology won the European Laboratory Research and Innovation Group’s Advances in Cell Based Screening in Drug Discovery Technology prize (6) and made The Scientistist’s list of top 10 innovations for the year (7). This technology leverages the small size and bright signal of NanoLuc® Luciferase to create a protein complementation system. With the NanoBiT® Technology, the NanoLuc® Luciferase is broken into two subunits, Large BiT (LgBiT) and Small BiT (SmBiT), that are optimized to have low affinity for each other. These two subunits can be chemically attached to molecules of interest. When the labeled molecules interact, the two subunits are brought close together and form the functional NanoBiT® luciferase—producing light when a detection solution is added.
Although NanoBiT® was first developed as a tool to monitor and measure protein:protein interactions (PPI), the bright signal, inherently low background and fast, add-incubate-read workflow make this luminescent technology ideal for immunoassay applications.
Recently Promega announced the release of a CE-marked in vitro diagnostic test for SAR-CoV-2 antibodies that is based on the NanoBiT® Technology. A version of the Lumit™ Dx SARS-CoV-2 Immunoassay has been available in the US since August 2020 under a policy allowing developers of serological tests to make them available while awaiting US Food and Drug Administration (FDA) review. The Lumit™ Dx SARS-CoV-2 Immunoassay detects SARS-CoV-2 antibodies in serum or plasma samples using a SARS-CoV-2-specific protein (CoV-2 protein) labeled with the two subunits of NanoBiT® (SmBiT or LgBiT). When these two labeled proteins are added to a sample that contains SARS-Cov-2 antibodies, they bind to the antibody, bringing the LgBiT and SmBiT subunits close together. The two subunits then form a functional bioluminescent enzyme and generate a luminescent signal in the presence of a detection reagent.
The novel bioluminescence detection method used by the Lumit™ Dx SARS-COV-2 Immunoassay enables the specific interaction between antibodies and SARS-CoV-2 proteins to be detected directly, without the intermediate steps required with other technologies like ELISAs. Removing these intermediate detection steps makes the workflow is simple and fast, going from sample to result in approximately one hour. Luminescent detection also eliminates the background signal, non-specific interactions and inconsistency that have plagued the immunoassay field.
As the world continues to develop tools to respond to the COVID-19 pandemic, antibody testing, alongside molecular tests, is playing a critical role. The availability of specific and sensitive immunoassays such as the Lumit™ Dx SARS-CoV-2 Immunoassay is important to accurately demonstrate the presence of antibodies to the virus (8). This ability is vital, not only for informing about individual exposures, but also to aid in population surveillance and to help with vaccine development efforts.
- Hall, M. P. et al. (2012) Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem. Biol. 7, 1848–57.
- Dixon, A. et al. (2016) NanoLuc Complementation Reporter Optimized for Accurate measurement of Protein Interactions in Cells. ACS Chem Biol. 11, 400–8. https://pubs.acs.org/doi/10.1021/acschembio.5b00753
- Machleidt, T. et al. (2015) NanoBRET–A Novel BRET Platform for the Analysis of Protein-Protein Interactions. ACS Chem Biol. 10, 1797–1804.
- Robers, M.B. et al. (2015) A luminescent assay for real-time measurements of receptor endocytosis in living cells. Anal. Biochem. 489, 1–8.
- Tran, V. et al. (2013) Highly sensitive real-time in vivo imaging of an influenza reporter virus reveals dynamics of replication and spread. J. Virol. 87, 13321–9.
- European Laboratory Research and Innovation Group (ELRIG) – advances in cell based screening in Drug Discovery meeting 2015: Best Technology (https://elrig.org/advances-in-cell-based-screening-in-dug-discovery-2015-technology-prize/ )
- The Scientist: Top10 Innovation 2015 (https://www.the-scientist.com/features/top-10-innovations-2015-34435 )
- Larsen, N. (2020)A Specific and Sensitive Matter: The Trouble with COVID-19 Antibody Tests. Promega Connections