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ATP Quantitation in the LMax Microplate Luminometer (MaxLine Application Note #40)

to 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.


REACTIONS PROFILES
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.


SUMMARY
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.


REFERENCES
1. Jones, D. 1998. Bioluminescence assays usi
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