Reliable Measurement of HTRF Assays in 1536-Well Format using Microplate Reader PHERAstar FSX

Introduction

In the pharmaceutical industry, it is a standard practice to screen new candidate drugs, but at the same time, requirements on instrumentation and assays are ever increasing. Especially, as there is a need to further increase the number of samples that can be measured in a day.

Using highly condensed microplates, for example 1536-well microplates, can help meet these requirements. The equipment of choice must have the ability to measure these microplates with the fastest possible read time, while achieving the assay performance criteria such as Z´ value or S/B.

The microplate reader PHERAstar FSX from BMG LABTECH can address the requirements for the ultra-high throughput screening process. Thanks to the new TRF laser, the fastest read times can be achieved in 1536-well microplates. This article demonstrates the enhanced performances that can be achieved with the PHERAstar FSX, using two HTRF assays from Cisbio Bioassays.

Assay principle

The levels of myo-Inositol 1 phosphate (IP1) in cultured cells can be directly detected using the HTRF IP-One terbium cellular assay. IP1 will be produced by cells following a ligand binding to a Gq coupled receptor. Using this assay, modulation of the Gq pathway by GPCR agonists and antagonists can be studied. Figure 1 shows the assay principle.

Figure 1. HTRF® IP-One terbium cellular assay principle.

Here, the HTRF donor (Lumi4™-Tb cryptate) is used to label a monoclonal antibody specific for IP1. The HTRF acceptor-labeled IP1 competes with intracellular produced IP1 for the antibody binding sites.

There is an inverse relationship between the specific HTRF signal and the cellular IP1 concentration in the sample. The protein binding domain-peptide interaction is detected by the second assay. Figure 2 shows the assay principle.

Figure 2. Histone – BRD4 interaction assay principle.

An anti GST antibody containing the HTRF donor will attach to a GST tagged bromodomain module (BRD4) and a biotinylated histone peptide is attached to the HTRF acceptor.

A high HTRF signal can be observed as long as the histone peptide interacts with bromodomain. The interaction will be prevented by inhibitors, such as (+) JQ1, causing a decrease in HTRF signal.

Materials and methods

  • EPIgeneousTM Histone/BRD4 Binding assay and HTRF IP-One assay from Cisbio
  • PHERAstar FSX microplate reader from BMG LABTECH featuring flash lamp and TRF laser
  • White 1536-well Hi-Base microplates from Greiner
  • Certus nano liquid dispenser from Gyger

All cell culturing and pipetting processes are in line with Cisbio’s recommendations1,2. Using the Certus liquid handler, the reagent is dispensed into 1536-well microplates. After allowing the microplates to sit for the necessary incubation period, they are read on the PHERAstar FSX by using the instrument settings listed in the below table.

Detection Method

Time resolved fluorescence (dual emission)

Optic module

HTRF

Ecitation

337

Emission A

665

Emission B

620

Integration start

60 μs

Integration time

400 μs

Light source

Flash Lamp or Laser

Ratio multiplier

10000

 

Results and discussion

IP-One cellular assay

The concentration dependent effect of atropine on the IP-One level is shown in Figure 3.

Figure 3. Antagonist atropine potency determination on CHO-M1 Gq coupled GPCR. Atropine inhibits the effect of a fixed concentration of acetylcholine agonist. Measurement was done on the PHERAstar FSX using the laser in flying mode (1 flash) to measure the HTRF signal. The 4-parameter fit was performed with the MARS Data Analysis software and was weighted using the formula 1/Y2.

The TRF laser was used in 1 flash flying mode to perform the measurements. The R2 value shown by the 4-parameter standard curve fit is 0.9994. Using the MARS Data Analysis software, the IC50 value calculated from the fit is 33 nM, which is in line with the expectations.

Histone-BRD4-interaction assay

Figure 4 shows the inhibiting effect of the known inhibitor (+) JQ1 on the histone-BRD4 interaction.

Figure 4. (+) JQ1 potency determination. Measurement was done on the PHERAstar FSX using the laser in flying mode (1 flash) to measure the HTRF signal. The 4-parameter fit was per- formed with the MARS Data Analysis software and was weighted using the formula 1/Y2.

The above figures demonstrate the ability of the PHERAstar FSX to reliably measure the HTRF assays in 1536-well format. The HTRF measurement using the TRF laser in flying mode will allow users to read the microplates within 36 seconds and achieve excellent Z´ value. The data also reveals that using the TRF laser will result in higher S/B values than flash lamp measurements (Figure 5).

Figure 5. Histone-BRD4 interaction assay. Comparison of Flash lamp and laser measurements for S/B, Z´value and Read times.

Conclusion

Rapid and reliable high throughput screening and compound follow up can be achieved when the PHERAstar FSX microplate reader is used in combination with Cisbio’s HTRF technology.

Acknowledgments

Produced from materials in originally authored by Franka Maurer1 ,Thomas Roux2 , Fanny Plénière2 and Najim Douayry2 from:

1 BMG LABTECH GmbH, Ortenberg, Germany

2 Cisbio Bioassays, Codolet, France

References

  1. http://www.cisbio.com/drug-discovery/ip-one-htrf- assay-kit
  2. http://www.cisbio.com/drug-discovery/epigeneoustm- binding-domain-assays

About BMG Labtech

BMG LABTECH is a leading developer and global manufacturer of microplate reader instrumentation with a wide range of measurement methods. Microplate readers are used in the pharmaceutical and biotech industries, as well as in academic research establishments, for both basic research analysis and High Throughput Screening. BMG LABTECH focuses solely on microplate readers and offers the most diverse selection of optical detection systems in conjunction with integrated liquid handling equipment.


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Last updated: Mar 8, 2017 at 9:55 AM

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