Evelyn McGown, Ph.D. and Michael Su, M.S.
Molecular Devices Corporation, 8/00
Adenosine triphosphate (ATP) is present in all living cells. Because the level isstrictly-controlled, assay of ATP can be used as a indicator of viable cell number.ATP measurements are used to monitor raw materials and manufacturing planthygiene for bacterial contamination in the food, drug, health care and personalhealthcare industries, as well as for waste water analysis.1 In the biotechnologyand pharmaceutical industries, ATP measurements are used to evaluate cellproliferation and cytotoxicity. The bioluminescent assay of ATP has become verypopular because of its high sensitivity and convenience. In this application note,we show the ATP assay results obtained with the Molecular Devices Lmaxmicroplate luminometer.
PRINCIPLE OF ASSAY
The basis for the assay is the reaction catalyzed by luciferase, obtained from the common American firefly (Photinus pyralis).2 The enzyme catalyzes the ATP-dependent oxidation of luciferin with the concomitant release of light (Figure 1).When ATP is the limiting component in the reaction, the amount of light emittedis proportional to the concentration of ATP. The intensity of the emitted light canbe measured easily with the Lmax microplate luminometer.
EQUIPMENT AND MATERIALS
1. Lmax microplate luminometer with SOFTmax PRO for Lmax (Molecular Devices Corp.)
2. ENLITENTM ATP Assay System, Cat. No. FF2000; Promega Corporation. 608277-2516. The kit contains Luciferase/Luciferin (L/L) reagent, ATP standard 10-7 M in HEPES buffer, pH 7.75) and ATP-free water.
3. Solid white 96-well microplates; e.g. Porvair PS white, E&G Wallac Cat. # 204003; Tel: 1-800-221-9650; or CorningCostar Cat. No. 3912; Tel: 1800492 1110). (In our hands, the Porvair PS plate has tended to have a lower background and slightly better sensitivity than the Costar plate.)
PREPARATION OF LMAX, REAGENTS AND STANDARDS AND ASSAY PROCEDURE
The instructions supplied with the kit (Technical Manual Part #TMF004)3 were followed, except for minor modifications to accommodate a 96-well format. NOTE: The technical manual also includes a section discussing sample preparation, including extraction of ATP from microorganisms or mammalian cells, which is not covered in this application note.
1. The M injector solvent delivery system was cleaned and sterilized by pumping 50% bleach through the injector and allowing it to stand for one hour, followed by rinsing with 50 mL deionized water. Then 75% ethanol was pumped through the system and allowed to stand for one hour, followed by a 100 mL deionized water rinse.
2. The enzyme and buffer solutions were thawed and kept on ice.
3. The L/L reagent was reconstituted by pouring the Reconstitution Buffer into the vial, gently swirling several times and allowing to stand at room temperature for one hour.
4. The solvent injection system was filled with L/L reagent by pumping 2.0 mL through it.
5. For fast kinetic assay to determine the best settings for endpoint assays, the Lmax was set to inject 100 uL (injector M) L/L reagent into selected samples of ATP dilutions (10 L/well) and then to immediately begin integrating at consecutive 0.1-sec intervals for a total of 10 seconds.
6. For determining sensitivity and dynamic range, the ATP standards were prepared by first diluting 100 L ATP stock standard with 900 L ATP-free water, followed by serial 1-to-10 dilutions in ATP-free water. The diluted standards (10 nM to 0.0001 nM) were kept on ice until use.
7. Diluted ATP standards (or ATP-free water blanks) were pipetted into micro-plate wells in triplicate (10 L/well). The final ATP content ranged from 0.01 to 1000 fmol/well.
8. For determining sensitivity and dynamic range, the Lmax was set up to inject 100 L/well, followed by a 2-second delay and a 1-second read.
9. The plate containing the ATP standards was placed into the drawer of the Lmax and the injection/read cycle initiated.
The reaction profile upon the addition of the luciferin/luciferase reagent is shown in Figure 2. The light emission in the presence of 100 fmol ATP/well (upper plot) was maximal within 1 seconds and remained at that level for at least 10 seconds. The blank (lower plot) showed essentially no response. Based on the data, we decided to use a delay of 2 seconds and then integrate total light for one second when reading an entire plate to determine sensitivity and dynamic range.
DYNAMIC RANGE AND SENSITIVITY
The standard curve from 0.01 to 1000 fmol/well is shown in Figure 3. The calculated limit of detection (amount producing a signal higher than 3 positive SD of the blank values above zero) was approximately 0.02 fmol/well. The dynamic range is at least 5 logs.
The Lmax microplate luminometer gives a detection limit of approximately 0.02 fmol ATP/well and a dynamic range of 5 logs with the ENLITEN ATP assay system. These results are as good as, if not better than, results obtained on a standard tube luminometer. The Lmax offers the advantage of a 96-well format and automated reagent addition for better precision and higher throughput. In addition, SOFTmax PRO for Lmax provides a powerful and convenient instrument control and data calculation package.
1. Jones, D. 1998. Bioluminescence assays using ATP. Luminescence Forum, 4: 1-9.
2. DeLuca, M.A. and W.D. McElroy, (1978) in Meth. Enzymol. vol 53:, p.3
3. Promega Corporation: www.promega.com
Note: Decontamination of the solvent delivery system is extremely critical for achieving satisfactory ATP assay results. Ethanol rinsing alone is not adequate. Not only must the system be sterilized, but any ATP remaining from killed bacteria must be destroyed in order to maintain blank values as low as possible.