Technical Note
Improve Crosstalk in Alpha Assays with Interlaced Reading
Teresa Castaño Martinez, PhD | Field Application Scientist | Molecular Devices
Introduction
Alpha technology including AlphaScreen® and AlphaLISA® assays from Revvity, is a bead-based technology that uses donor and acceptor beads that are conjugated to biomolecules of interest. When the biomolecules interact, the beads are brought into close proximity, enabling an energy transfer to produce a chemiluminescent signal that is measured using a microplate reader. Alpha technology is commonly used to study molecular interactions, with its advantages including (1) miniaturization, (2) high signal-to- background ratio, (3) homogeneous assay format and (4) the ability to multiplex, all of which make this technology suitable for high-throughput assays. However, the generation of a very bright signal can lead to an increase in well-to-well cross-talk. Therefore, the selection of a suitable microplate reader and experimental design can help decrease crosstalk, potentially improving the quality of the data.
This technical note focuses on the advantages of using the Interlaced Reading feature in SoftMax® Pro Data Acquisition and Analysis Software to decrease crosstalk in Alpha assays. These assays are performed on the SpectraMax i3x Multi-Mode Microplate reader together with the AlphaScreen cartridge.
Molecular Devices Alpha assay - compatible microplate readers
AlphaScreen and AlphaLISA signals can be read on SpectraMax® i3x Multi-Mode Microplate Reader and SpectraMax Paradigm Multi-Mode Microplate Reader together with one of the following detection cartridges:
- AlphaScreen 384 Std Detection Cartridge
- AlphaScreen 384 HTS Detection Cartridge
- AlphaScreen 1536 HTS Detection Cartridge
The choice of the detection cartridge will depend on your plate format and desired throughput. In the following table the recommend cartridge are shown; the choice will depend on your plate format and typical throughput.
All the AlphaScreen Detection Cartridges use a laser diode for sensitive detection of Alpha assays. In addition, the cartridges contain an optimized design that isolates each well during detection, reducing crosstalk and enabling optimal performance for Alpha assays.
Table 1. Compatible plate formats and read time for the three types of AlphaScreen cartridges.
Reader setup recommendations
All Molecular Devices microplate readers are controlled by SoftMax Pro software, enabling consistency between readers and collaborators. For Alpha assays, the following instrument settings are recommended:
Read order: user selectable
Show Pre-Read Optimizations Options: Yes (microplate optimization)
Normalization: user selectable
Interlaced Reading: Yes
Table 2. Recommended instrument settings for AlphaScreen assays on the SpectraMax i3x reader. A pre-configured protocol called “AlphaScreen 384 HTS” is available in the protocol library.
What is cross-talk and how can interlaced reading decrease it?
Crosstalk is what happens when signal produced in one well of a microplate is not confined to that well and interferes with the signal in adjacent wells. This undesired effect is commonly present in Alpha assays due to
the long decay time. When a well is excited, the decay time of the Alpha signal is quite long and the emission of light may be longer than the read time of that well.
Therefore, when the detector is moving to the next well, the adjacent well that was just measured is still emitting light. Consequently, the measured light in the second well can be “contaminated” with light coming from the first well, producing what is commonly known as crosstalk.
One of the benefits of the SpectraMax i3x reader with SoftMax Pro software is the reduction of crosstalk using the Interlaced Reading option (Figure 1). Similar to a chessboard pattern, the instrument reads every second well (A1, A3, A5…) instead of reading adjacent wells consecutively (A1, A2, A3). The instrument reads every other well of the microplate, and then will return to read the skipped wells (A2, A4, A6…). This read pattern improves
the quality of the data, as shown for the standard curve (Figure 2), decreasing the %CV values, especially at low concentrations (Table 3). Crosstalk was calculated by comparing the nearest empty wells to the highest standard samples. The data shows a 74-fold reduction in crosstalk when the measurement was taken using Interlaced Reading (Figure 3).
Figure 1. Chessboard Pattern which represents how interlaced reading works on the SpectraMax i3x for AlphaTechnology.
Table 3. Comparison table showing mean counts and %CV values at low concentrated samples (0.032 mg/ml) when reading with interlaced settings ON vs. interlaced settings OFF.
Figure 2. Sample standard curves on the SpectraMax i3x reader showing the signal measured with (blue plot) and without (orange plot) interlaced reading. R2 = 1.0 with interlaced ON (n=2) vs. R2= 0.996 with interlaced OFF (n=2)
Figure 3. The upper graph shows the Counts read using the SpectraMax i3x reader in the highest standard wells and in empty wells using the interlaced read OFF vs. interlaced read ON. In the bottom graph, the crosstalk (%) was calculated for both reads. For that, the signal from empty wells was measured, and divided by the signal from the highest standard sample. The resulting crosstalk was reduced 74-fold when interlaced reading was used.
Conclusion
Together, these results demonstrate that using the interlaced reading option available with the SpectraMax i3x and Paradigm readers and SoftMax Pro software can reduce crosstalk significantly in AlphaScreen and AlphaLISA assays. In addition, the reduction in CV%s at the bottom of the standard curve has a beneficial effect on data fidelity and may allow researchers to detect lower concentrations of test molecules more accurately.