Franka Ganske, BMG LABTECH, Offenburg, Germany; EJ Dell, BMG LABTECH, Durham, USA; Brad Larson, BellBrook Labs, Madison, USA, 12/2008
BellBrook Labs has developed the universal Transcreener® ADP2 Assay, a homogeneous, competitive fluorescent polarization HTS assay that directly detects ADP, the invariant reaction product of all kinase reactions, as well as heat shock proteins and other ATPases. These enzymes catalyze the covalent regulatory reactions that are central to cell signaling and are high value targets in drug discovery. The Transcreener ADP2 FP Assay is a new assay, with greater sensitivity than the original Transcreener ADP Assay.(1) The improvement is a more sensitive antibody against ADP yielding an excellent signal at less than or equal to 10 % ATP consumption for a broad range of initial ATP concentrations (0.1-1,000 µM).
BMG LABTECH’s PHERAstar Plus (Figure 1) is a multidetection microplate reader that combines rapid plate reading necessary for HTS with enhanced performance and sensitivity needed to measure small liquid volumes. The PHERAstar Plus reads all HTS detection modes (fluorescence intensity, time-resolved fluorescence, fluorescence polarization, luminescence, and absorbance) in all plate formats up to 1536 wells.
The PHERAstar Plus uses a unique application-specific module in conjunction with an optical reading head featuring two matched pairs of photomultiplier tubes (PMTs) that can simultaneous measure two emission signals at any desired wavelength. FP, TR-FRET and BRET are powerful detection modes that benefit from the Simultaneous Dual Emission technology. The outstanding sensitivity of the PHERAstar Plus is based on a new, innovative lens-based optical design. This design provides for outstanding sensitivity and accuracy, along with minimal read times. For FP assays, capturing both the parallel and perpendicular channels with one read dramatically decreases the inherent variability in reading the same plate twice.
Fig. 1: BMG LABTECH's multidetection HTS microplate reader PHERAstar Plus
The Transcreener® ADP2 Assay is a fluorescence polarization immunoassay based on the detection of ADP by an antibody (Figure 2). This assay platform provides the possibility to universally interrogate all enzymes that catalyze group transfer reactions with ATP. In step one of the assay, enzymes catalyze the transfer of phosphate from ATP to a protein, peptide, lipid or small molecule resulting in the accumulation of ADP.
Fig. 2: Transcreener ADP2 Assay Principle for Kinases
In step two the Transcreener® ADP2 Detection Mixture, which contains an ADP Alexa633 tracer bound to an anti-ADP antibody, is added. If there is enzymatic activity resulting in necessary ADP then the bound tracer is displaced by the ADP. The free tracer rotates quickly leading to a lower polarization value. If there is no free ADP because of no enzymatic activity, the tracer is still bound to the antibody. This whole construct rotates very slowly giving a higher polarization number. Therefore, ADP production leads to a decrease in fluorescence polarization.
Using 10 µM ADP and 10 µM ATP stock solutions a 12 point ADP/ATP standard curve was prepared, while keeping a constant concentration of total adenosine.
This standard curve mimics a kinase or ATPase reaction (Note ADP is produced while ATP is depleted). The upper limit of the standard curve was set to 0 µM ADP/10 µM ATP (mimicking 0% conversion) and the lower limit was set to 10 µM ADP/0 µM ATP (mimicking 100% conversion). To the different ADP/ATP solutions the same volume of ADP Detection Mixture was pipetted. The antibody concentration was 7.4 µg/mL. (For ideal assay performance it is important to determine an optimal antibody concentration under the specific enzyme and buffer conditions used in your experiment).(2) The concentration of the far-red tracer was 2 nM. The solutions (384 well final volume 20 µL, 1536 well final volume 8 µL) were mixed and incubated for 1 hour at room temperature. The fluorescence polarization measurements were performed using the Transcreener® specific FP optical module with Excitation at 590 nm and Emission A (parallel) and Emission B (perpendicular) at 675 nm. The mP target was set to 20 mP for the free tracer.
Figure 3 and 4 show the standard curves measured on the PHERAstar Plus in 384 well and 1536 well format, respectively. Graphing on the log scale eliminates the point that corresponds to zero. To include all twelve points along the curve, the value for 0 µM ADP/10 µM ATP was graphed at 0.01 µM position.
Fig. 3: ATP/ADP standard curve performed in a 384 well microplate
Fig. 4: ATP/ADP standard curve performed in a 1536 well microplate
Both graphs show similar assay windows and also similar EC50 values indicating that the Transcreener® ADP2 assay can be performed on the PHERAstar Plus using both plate types.
In order to show that the new Transcreener® ADP2 assay is favorably compared to the former Transcreener® ADP assay, a Z’ and ΔmP comparison between both assays was performed (Figure 5).
Fig. 5: Z’and ΔmP comparison between the Transcreener® ADP and ADP2 FP assays.
Z’validation minimal qualification is shown by the red dashed line. ΔmP validation
minimal qualification is shown by the black dashed line.
The lowest % ATP conversion level yielding a Z’ > 0.7 is obtained at 0.8 µM ADP (or 8 % ATP conversion) for the ADP assay (purple line) whereas for the ADP2 assay already at 0.4 µM ADP (or 4 % ATP conversion) the intended Z’ > 0.7 is reached (blue line).
The results show that the Transcreener® ADP2 FP assay is able to yield higher quality data at conversion levels that lie within initial rate enzyme reaction kinetics.
The universally generic nature of the Transcreener® ADP2 kit will reduce assay development efforts thus allowing HTS to occur earlier. As a characteristic parameter for the quality of the assay, a Z' value > 0.7 was calculated, which represents an excellent assay performance. Z' values between 0.5 and 1 indicate a highly robust screening assay and reflect high quality of instrumentation.(3)
The data show that the PHERAstar Plus passes the validation criteria from BellBrook Labs. The PHERAstar Plus microplate reader provides the ideal platform for the Transcreener® ADP2 Assay. With its dual wavelength emission detection and five photomultiplier tubes (PMTs), the PHERAstar Plus provides the speed and sensitivity needed to take full advantage of BellBrook Labs Transcreener® technology. Furthermore, BMG LABTECH has designed an optic module specifically for BellBrook Labs’ Transcreener®, thereby making assay setup simple.
Transcreener® is a patented technology of BellBrook Labs.