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A highly sensitive ATP-luminescence assay using VICTOR2 with the dispenser option

INTRODUCTION

In high sensitivity flash type luminescence assays the measurement is done just after dispensing the activating reagent or after a short delay time. Over the past years luminescence substrates have been improved towards providing more stable signal. These so-called glow type substrates allow microplates to be counted without dispensors. However, flash type reagents will always have one advantage, sensitivity. In the glow type the luminescence signal is spread over a wide time scale (for example half life 30 min) and only a portion of it is read (1 - 10 s / well), so a lot of light is not detected. In flash assays most of the light is counted (half-life 5 s).

Adenosine triphosphate (ATP) is the energy reserve essential for viable cells. The concentration of ATP is stable in cells. Therefore the amount of ATP in the well correlates with the number of viable cells. ATP assays can be used for cell proliferation, cytotoxicity and cell adhesion studies. One application is the detection of microbial contamination in milk. In this study ATP samples were assayed with a Boehringer Mannheim flash type kit using VICTOR2 dispenser option. The detection is based on a light emitting (560 nm) reaction of ATP and luciferin in the presence of luciferase enzyme and oxygen.

METHODS

The assay of an ATP dilution series in a plate with a constant luciferase concentration was performed according to the kit instructions (ATP Bioluminescence Assay kit HS II, Boehringer Mannheim, Germany). 50 μL of Luciferase reagent was dispensed into white microplates (Combiplate, Labsystems, Finland) with 25 μL of ATP sample and 25 μL of cell lysis reagent. The wells were measured for 10 seconds after a 1 s delay.

RESULTS

The dilution series proved to be linear over 5 decades with a detection limit of 1.0E-11 M ATP, see figure 1. In the consistency measurement the intra-plate CV% was 3.2, with minimum and maximum differing only by 14% and 7% respectively from the average 21173 cps.

CONCLUSION

VICTOR2 with the dispenser option is an ideal instrument for microplate assays requiring immediate measurement after reagent injection. Applications are not restricted to luminescence assays but also cover fluorescence assays like Ca2+ ion channel studies.


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