Applications

We find new applications for the technology that is the utilization of firefly reaction every day. Below you’ll find the major categories that we sell to under the BioThema brand.

All ATP producing and ATP degrading enzymatic reactions can be continuously monitored by measuring the light from firefly luciferase/luciferin being present in the reaction mixture. Examples include protein kinases⁶⁷, adenylate kinase¹⁴, pyruvate kinase⁶, pyruvate phosphate dikinase, glycerol kinase^{43, 44}, creatine kinase isoenzymes^{8, 10, 18, 22}, ATPases¹⁴, ATP synthetases⁴⁹, aminoacyl tRNA synthetase. Substrates to such enzymes can be measured kinetically or with endpoint assays. Examples include ADP, AMP, pyrophosphate. When continuously monitoring ATP the luciferase ATP depletion should be negligible compared to the enzymatic reactions being measured.  ATP concentrations from 1 to 1,000,000 pmol/L can be monitored. ATP depletion assays are best set up as 1^st order reactions plotting the natural logarithm of the light versus time. The slope in such plots is the rate constant of the reaction. ATP forming reactions can be calibrated by measuring the light before and after adding a known amount of ATP standard at the end of each test.

All living cells contain ATP and when the cell dies ATP is degraded by intracellular enzymes to ADP and finally AMP. The intracellular ATP concentration is similar in different cell types. This means that bacterial cells contain 1-2 attomoles per cell while animal and plant cells contain 100 to 10 000 times as much ATP. Extracellular ATP may be degraded by adding ATP consuming enzymes or by physical separation. Determination of intracellular ATP can then be used to estimate biomass. If the amount of ATP per cell is known the cell number may also be estimated. In sample which like urine may contain, extracellular ATP, ATP in somatic cells and ATP in bacterial cells all three ATP pools can be determined. Determination of bacterial ATP in urine is used to determine bacteriuria in the diagnosis of urinary tract infection. All ATP assays shall be calibrated by measuring the light before and after adding a known amount of ATP standard at the end of each test. This calibration obviates analytical interference from all sorts of variation of reagents and sample matrices and provides a result expressed in moles rather relative light units.

The luc gene coding for firefly luciferase may be attached to another gene of interest. The expressing of this gene may then be estimated from the level of luciferase expression measured as light. This may be done in vitro, i.e. after lysing the cells adding ATP and D-luciferin, or in vivo. In the latter case D-luciferin has to enter the cells. Even whole live animals or plants may be studied, “in vivo imaging”, by injecting D-luciferin into the organism (ATP is already in the cells).

All biological residues contain ATP. When living cells die ATP is degraded to ADP and finally AMP. Measurement of ATP+AMP is a proof of biological residues being present. This can be done using the unique Kikkoman LuciPac Pen (surfaces) or LuciPac Pen Aqua (liquids) with a portable instrument PD-30. Hygiene control measuring ATP only may underestimate the contamination if ATP has been degraded in the biological residues. On the other hand ATP is an indicator that the residues are still alive. Neither ATP nor ATP+AMP are a reliable measure of bacteria as these contain much less ATP than cells from animals or plants. Assays can be calibrated by relating the light from the sample to a known amount of certified liquid-stable ATP standard. In case one wants to use the ATP assay to measure living bacteria the surface or liquid must first be treated with the ATP Eliminating Reagent to degrade extracellular ATP.

Antibodies or antigens may be labelled with luciferase or ATP producing enzymes.  Biotinylated recombinant luciferase can be detected at 10^-20 mol and biotinylated acetate kinase at 10-^21 mol.

BioThema is constantly getting requests about developing customized assays and we have been doing just that for more than 40 years. Usually is a matter of setting up new assay protocols for our already available kits but sometimes there’s a need for new reagents also. BioThema is a long time OEM manufacturer of ATP reagent and would be glad to help you make an assay that fits your need.