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Cathy Olsen, PhD | Sr. Applications Scientist | Molecular Devices

Introduction

This application protocol describes the optimal instrument parameters used to validate the SpectraMax® Paradigm® Multi-Mode Microplate Reader with the following assays from BellBrook Labs:

• Transcreener ADP2 FP (3010)
• Transcreener AMP/GMP (3006)
• Transcreener UDP FP (3007)
• Transcreener GDP FP (3009)

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 these enzymes catalyze the covalent regulatory reactions that are central to cell signaling and are of great value as targets in drug discovery.

The Transcreener FP Assays are a single step, competitive immunoassay for direct detection of nucleotides with a far red fluorescence polarization (FP) readout. The reagents for all of the assays are a far red Tracer bound to a highly-specific monoclonal/polyclonal antibody. Nucleotide diphosphate or monophosphate produced by the target enzyme displaces the tracer from the antibody, leading to increased rotational freedom and results in a decrease in polarization (Figure 1). The use of a far red tracer minimizes interference from fluorescent compounds and light scattering. The Transcreener FP Assays are designed specifically for HTS with a single addition, mix-and-read format.

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 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 is decreased proportionately, maintaining a total adenine nucleotide concentration of 10 μM. The integration times were varied to determine the requirements for a Z’ > 0.5. In order to validate an instrument for use with the Transcreener FP Assays a Z’ > 0.7 and a Δ mP > 120 at 10% conversion of 10 μM ATP were required.

Materials

• ATP/ADP Mixture – 4 mM MgCl2, 2 mM EGTA, 50 mM HEPES, pH 7.5, 1% DMSO, 0.01% Brij-35, and ATP/ADP (combined to a constant adenine concentration of 10 μM)
• ADP Detection Mixture – 1X Stop & Detect Buffer B, 4 nM ADP Alexa633 Tracer, and 14.8 μg/mL ADP2 Antibody
• Free Tracer – 1X Stop & Detect Buffer B and 4 nM ADP Alexa633 Tracer
• Buffer Blank – 1X Stop & Detect Buffer B and 14.8 μg/mL ADP2 Antibody

For a detailed procedure on how to prepare a standard curve, please refer to the appropriate Transcreener Technical Manual (http://www.bellbrooklabs.com/ transcreener_hts_assays.html).

Figure 1. Transcreener FP assay principle. The SpectraMax Paradigm Multi-Mode Microplate Reader offers dual PMTs for faster detection of FP signal and has a modular design with assay-specific cartridges for optimal performance. It supports up to 1536-well microplate formats and is HTS-compatible. With optimized settings for the Transcreener FP assays, the SpectraMax Paradigm reader exceeds BellBrook Labs validation requirements.

Methods

Assay preparation

Step 1. Dispense 10 μL of each ATP/ADP combination across an entire row of a 384-well plate.

Step 2. Add 10 μL of ADP Detection Mix to those rows.

Step 3. Dispense 10 μL of the 10 μM ATP/0 μM ADP combination into row P.

Step 4. Dispense 10 μL of Free Tracer into wells P1-P12.

Step 5. Dispense 10 μL of Buffer Blank into wells P13-P24.

Instrument setup

Set up the SpectraMax Microplate Reader with the settings in Table 1.

Table 1. Recommended SpectraMax Paradigm Instrument settings

The SpectraMax Paradigm plate reader is operated using SoftMax® Pro Software. Start with a pre-configured protocol from the protocol library and modify as described.

Step 1. Open the SoftMax Pro Software and click on ‘Protocol Manager’, then ‘Protocol Library’, to access the pre-configured protocols. In the ‘Paradigm Protocols’ folder, select the ‘FP Rhodamine’ protocol.

Step 2. Click ‘Plate01’ in the Navigation Tree, and click on Settings (gear icon) to open the Settings dialog.

Step 3. Select the custom cartridge for Alexa Fluor 633 FP in the Cartridges menu.

Step 4. Read Mode ‘FP’ and Read Type ‘Endpoint’ should be highlighted.

Step 5. Wavelength settings are determined by the cartridge and are not user-changeable.

Step 6. Click ‘Plate Type’ and choose Plate Format ‘384 Wells’ and Select Specific ‘384 Well Corning low vol/rndbtm’.

Step 7. Click ‘Read Area’ and choose the wells to read on the plate diagram.

Step 8. Click ‘PMT and Optics’. Select ‘Off – Stop and Go’ or ‘Performance – On the fly’.

Step 9. If Stop and Go is selected, enter an integration time of 20 ms or higher. Longer integration times typically offer better performance, but plate reading will take longer.

Step 10. If optimal read height is known, enter it. For any new assay or plate type, read height optimization should be performed. If optimization will be performed, leave the read height at the 1 mm default value. After optimization is performed, the new (optimal) read height will replace the default value.

Step 11. Click ‘More Settings’. Choose a read order, ‘Row’ if a whole plate or a few complete rows will be read, or ‘Column’ if only a few complete columns will be read.

Step 12. Check the box next to ‘Show Pre-Read Optimization Options’.

Step 13. Click ‘OK’ to close the Settings dialog.

Step 14. Click Read. A wizard showing optimization options will appear. Perform both microplate optimization and read height optimization for any new assay.

Figure 2. Assay validation parameters. A: Z’ and Δ mP values observed in a standard curve mimic conversion of 10 μM ATP to ADP. B: Zoomed view of the 0-3 μM ADP section of the standard curve shows the Z’ validation minimal qualification data (red dotted line) and Δ mP validation minimal qualification data (black dashed line). The 10% ATP conversion validation point is also indicated (vertical black dotted line). Plate reader set at 100 ms integration time.

Table 2. Assay performance with various instrument settings

Results

Sample FP standard curve

As the ratio of ADP to ATP increases, the proportion of bound tracer vs. free tracer decreases, resulting in an overall decrease in mP values. Assay plates containing the 15-point standard curve were read on a SpectraMax Paradigm reader.

Conclusion

This application protocol demonstrates the validation of the SpectraMax Paradigm reader for use with the Transcreener FP Assays. By utilizing the optimized instrument settings suggested here, Z’ values > 0.7 and Δ mP > 120 are achievable with read times under 1 minute. Results greatly exceeding minimum validation requirements can be achieved by using longer integration times, while the faster on-the-fly setting meets assay requirements with a read time of less than one minute.

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