Optimized settings for Transcreener TR-FRET assays on SpectraMax iD5 and i3x readers

Joyce Itatani | Applications Scientist | Molecular Devices

Cathy Olsen | Sr. Applications Scientist | Molecular Devices

Introduction

Transcreener® HTS is a universal, high throughput biochemical assay platform based on the detection of nucleotides, which are formed by thousands of cellular enzymes—many of which catalyze the covalent regulatory reactions that are central to cell signaling and are of great value as targets in drug discovery.

The Transcreener® TR-FRET Assays are single step, competitive immunoassays for direct detection of nucleotides with a far-red time-resolved Förster resonance energy transfer (TR-FRET) readout. The reagents for all of the assays are a far-red tracer bound to a highly specific monoclonal antibody-terbium conjugate. Excitation of the terbium complex in the UV range (ca. 330 nm) results in energy transfer to the tracer and emission at a higher wavelength (665 nm). Nucleotide diphosphate or monophosphate produced by the target enzyme displaces the tracer from the antibody, leading to a decrease in TR-FRET (Figure 1). The use of a red tracer minimizes interference from fluorescent compounds and light scattering. The Transcreener TR-FRET Assays are designed specifically for high-throughput screening (HTS) with a single addition, mix-and-read format.

Figure 1. Transcreener TR-FRET assay principle. This competitive assay uses a donor-labeled antibody and a fluorescently labeled tracer. When no analyte is present, FRET occurs. In the presence of analyte, the tracer is competed off the antibody, and FRET is disrupted.

Validation criteria

A critical factor in realizing the advantages of the Transcreener HTS assays is the correct setup of the microplate reader used for data readout. Proper selection of instrument settings, including filters and other components, can impact the instrument’s sensitivity for any given assay. The key instrument parameters for Transcreener HTS assay performance were identified by running a 10 μM ATP/ADP standard curve (24 replicates), as standard curves of this type mimic enzyme reactions. Starting with 10 μM ATP, ADP was added in increasing amounts and ATP was decreased proportionately, maintaining a total adenine nucleotide concentration of 10 μM. The flash numbers were varied to determine the requirements for a Z’ > 0.5. An instrument is validated for use with Transcreener TR-FRET assays if a Z’ > 0.7 at 10% conversion of ATP is achieved.

Materials

• Transcreener ADP² TR-FRET Red Assay (cat. #3011-1K)
• ATP/ADP Mixture: 10 mM MgCl₂, 20 mM HEPES, 0.01% Brij-35 and ATP/ADP (combined to a constant adenine concentration of 10 μM)
• ADP Detection Mixture: 1X Stop and Detect Buffer C, 8 nM ADP2 Antibody-Tb Conjugate, 26.8 nM ADP HiLyte 647 Tracer
• High FRET Mixture: 8 nM ADP² Antibody-Tb Conjugate, 26.8 nM ADP HiLyte 647 Tracer, 1X Stop and Detect Buffer C, 10 μM ATP
• Low FRET Mixture: 8 nM ADP² Antibody-Tb Conjugate, 26.8 nM ADP HiLyte 647 Tracer, 1X Stop and Detect Buffer C, 10 μM ADP

Standard curve setup

1. Dispense 10 μL of each ATP/ADP combination across the entire row of a 384-well plate.
2. Add 10 μL of ADP Detection Mixture to those rows.
3. Dispense 10 μL of the 10 μM ATP/0 uM ADP combination into row P.
4. Dispense 10 μL of High FRET Mixture into wells P1-P12.
5. Dispense 10 μL of Low FRET Mixture into wells P13-P24.

For a more detailed procedure on preparation of a standard curve, please refer to the appropriate Transcreener Technical Manual (https://www.bellbrooklabs.com/technical-resources/technical-manuals/).

Microplate readers

• SpectraMax® iD5 Multi-Mode Microplate Reader (Molecular Devices cat. #ID5-STD), equipped with the following:
  • Enhanced TRF Module (Molecular Devices cat. #0200-7030)
  • Filter 320/100 (available from Molecular Devices)
  • Filter 616/10 (Molecular Devices cat. #6590-0118)
  • Filter 665/10 (Molecular Devices cat. #6590-0121)
• SpectraMax i3x® Multi-Mode Microplate Reader (Molecular Devices cat. #I3X), equipped with the following:
  • HTRF Detection Cartridge (Molecular Devices cat. #0200-7011)

Instrument setup

The SpectraMax iD5 and SpectraMax i3x readers use SoftMax® Pro Data Acquisition and Analysis Software. Proceed with the following steps using optimized settings.

1. Open the SoftMax Pro Software and select TR-FRET for the Read Mode and Endpoint for the Read Type. On the SpectraMax i3x reader, HTRF (cartridge) should be selected as the Optical Configuration.
2. On the SpectraMax iD5 reader, install the 320/100 excitation filter and 616/10 and 665/10 emission filters, and then select these filters from the Excitation and Emission dropdown wavelength menus. On the SpectraMax i3x reader, install the HTRF cartridge, and wavelengths will be selected automatically when HTRF is selected as the Optical Configuration.
3. Plate Type and Read Area should be selected based on the microplate used and wells to be read. For the standard curve described, all wells of the microplate are read.
4. PMT and Options Settings: Set Flashes per Read (or Pulses per Read) to 20 to 100 (for results shown here, 50 was used), measurement delay to 0.05 ms, and integration time to 0.7 ms.
5. Microplate and read height should be optimized for each new lot of microplates or assay volume used.

The same instrument settings can be used to read subsequent plates as long as the volumes, microplate lot, tracer, and concentrations remain the same. Table 1 summarizes the settings described above.

Table 1. Recommended settings for SpectraMax iD5 and i3x readers. Microplate and read height should be optimized for each new lot of microplates or assay volume used (use the Show Pre-Read Optimization options in the instrument settings in SoftMax Pro Software).

Conclusion

We demonstrate the validation of SpectraMax iD5 and SpectraMax i3x readers for use with the Transcreener ADP2 TR-FRET Red Assay. By using the optimized instrument settings, Z’ values > 0.8 are achievable (Table 2). For both instruments, Z’ values above the validation criterion of 0.7 were reached at ATP conversion rates of 2% or more (Figure 2), exceeding the minimum validation requirement of a Z’ greater than 0.7 at 10% ATP conversion.

Table 2. Assay performance with different reader settings

Figure 2. A) Z’ values observed in a standard curve mimic conversion of 10 μM ATP to ADP. B) Zoomed view of the 0–2 μM ADP section of the standard curve shows the Z’ validation minimal qualification data (red dotted line) and 10% ATP conversion validation point (black dotted line). Blue plots show data from the SpectraMax iD5 reader, and green plots show data from the SpectraMax i3x reader, both with 50-ms integration time.