BioThema is entirely focused on analytical applications of the firefly luciferase reaction whether it is detecting cells, measuring enzyme activity or something else. In this reaction luciferin is activated with ATP (adenosine triphosphate) and thereafter oxidized to oxyluciferin and CO2. The oxyluciferin is formed in an excited state and emits light when going to its ground state. The light is normally measured in what is called a luminometer.
In assays of ATP the light emission may be essentially constant or decay rapidly. Thus there are stable light and flash type ATP reagents based on the firefly luciferase reaction. At low levels of luciferase a negligible proportion of the ATP is consumed per minute and the light can then be almost constant but also low. At high levels of luciferase it is inevitable that ATP is consumed resulting in a rapidly decreasing light but initially the peak light is high. Actually the peak height and the decay rate are both proportional to the luciferase activity. BioThema has developed three types of ATP reagents66:
- ATP Reagent SL (stable light) with a decay rate <1 %/min (very similar to the no longer available ATP Monitoring Reagent from Bio-Orbit Oy). When these reagents are added to a reaction mixture where ATP is formed or degraded the ATP concentration can be continuously monitored by measuring the light emission 9, 10, 21, 22, 32, 34, 43, 44, 49, 64, 66, 67, 69, 72. ATP Reagent SL can also be used to measure ATP in cultivated eukaryotic cells containing e.g. 20 fmol/cell as the linear range of the assay is 1-50.000 cells31, 36, 37, 40, 59.
- ATP Reagent HS (High Sensitivity) with a decay rate around 10 %/min. This reagent contains 20x the activity of ATP Reagent SL and is mainly intended to detect low levels of bacteria. Bacterial cells contain around 1 amol ATP per cell. With ATP Reagent HS 10 amol ATP can be detected corresponding to around 10 bacterial cells. With a good luminometer the linear range is 10-100.000 amol ATP. This reagent can be manually injected in the sample as placing the cuvette in the luminometer and measure the light signal only takes a few seconds72.
- ATP Reagent SS (Super Sensitive) with a decay rate around 235 %/min and contains 235x as much luciferase as ATP Reagent SL. After 1 minute only 10 % of the light remains. This means that it is necessary to inject the reagent into the sample to catch the signal. The detection limit with this reagent is <1 amol ATP (Berthold Detection Systems, Application Note 2010/10. At such ATP levels one finds ATP contaminations on pipette tips, microplates and everywhere. This reagent should only be used when a really high sensitivity is required72.
The light signal is usually expressed in Relative Light Units (RLU). These units vary between instruments, reagents and sample matrices and with temperature72. It is therefore necessary to calibrate each assay by measuring the light signal before and after adding a known amount of ATP Standard. BioThema has a series of certified liquid-stable ATP Standards68. They should be added in a small volume (10 µL) not to change the reaction conditions. The amount should be at least 10x the amount of ATP from the sample. The use of ATP standards in this way compensates for all types of analytical interference and correct results are always obtained. This is not the case if you refer to an ATP standard curve as there can be interference from the sample matrix and variations in sample temperature72.
The above ATP reagents and ATP standards are combined with various other reagents to a number of kits for special purposes. These reagents include:
- ATP Eliminating Reagent for degrading extracellular ATP when determining intracellular ATP.
- Cell Lysing Reagent for lysing somatic cells leaving bacterial cells unaffected.
- Extractant B/S for releasing adenine nucleotides from bacterial and somatic cells.
- Luciferin Substrate and ATP Substrate for assays of firefly luciferase.
- D-luciferin (free acid, potassium salt and sodium salt) for in vivo imaging71.
We also manufacturer customized reagents and kits for other companies (for freeze dried ATP reagents the minimum order is 800 vials).
Recommended overview papers:
Lundin, A. (2000) Use of Firefly Luciferase in ATP-Related Assays of Biomass, Enzymes, and Metabolites. Methods in Enzymology, 305, 346-370.
Lundin A. (2014) Optimization of the firefly luciferase reaction for analytical purposes. Adv. Biochem. Engin./Biotechnol. 2014; 145:31-62”