Miniaturising screening assays: a case study

Speaker: Céline Legros (Project Manager Screening Assay Development, Servier, Paris), Catherine Wark (Applications Manager, BMG LABTECH Ltd.)

Drug screening studies are often limited by sample amount and time restrains. Screeners need to find the right balance between saving time and money on one hand and producing reliable, reproducible, and quantitative results on the other. Therefore, approaches that minimize sample consumption and time while still delivering high-quality hits are constantly looked for.

In high-throughput screening (HTS), the measurement of the biological responses is usually performed by a microplate reader. Consequently, the selection of the best-suited plate reader has a major impact on the success of screening campaigns. Indeed, sensitivity and speed of detection of the instrument exert a positive impact on data quality and cost savings and hence are of major relevance for successful screening campaigns.


Sensitivity helps saving money and time

Although sensitivity directly affects data quality, it indirectly affects screening costs as well. Because of the plate reader´s sensitivity, assays can be migrated from 384- to 1536-well plates or expensive reagents can be further diluted, while keeping the same plate format. This obviously results in cost savings.

In addition, sensitivity positively affects the speed of detection. For instance, on a highly sensitive microplate reader, the number of excitation flashes can be reduced without negatively affecting data quality. Keeping good Z´ values with a reduced flash number for each sample well can generate considerable time savings when multiplied by 384 or 1536, and further by the number of plates run in a day or for an entire campaign.


Assay downscaling

The miniaturisation or downscaling of a screening assay represents a key step in the drug discovery process. As the term suggests, the goal of miniaturisation is the reduction of sample volumes and formats in order to save on reagents and time, while maintaining reliability, robustness, and reproducibility.

Miniaturisation can be applied to different detection modes and purposes. Apart from drug screening, even protein concentration determination could be downscaled for proteomic applications, as for this BCA protein quantification assay downscaled to 384- and 1536- well plates.

In addition, miniaturisation has to support the implementation of the most efficient automated process still able to deliver quality hits. For instance, sample usage can be limited to 2 µL in 1536-well plates when a PHERAstar plate reader is combined with dedicated liquid handling devices as shown in the miniaturization of a cell-based TNF-α AlphaLISA assay.

Although 384- and 1536-well formats are the most commonly used in HTS, assays could even be downscaled to 3456-well plates as shown in the miniaturization of cell-based GPCR and kinase assays.

Servier: a case study

In this free scientific talk here below, we present a case study where the use of the PHERAstar FSX high-throughput plate reader significantly contributed to the miniaturisation of two screening assays at Servier.

Servier’s screening department is equipped with several BMG LABTECH readers. A CLARIOstar, a NEPHELOstar, and three generations of PHERAstars are currently used for primary and secondary drug discovery campaigns either as standalone instruments, coupled with stackers, or fully integrated into medium- and large size robotic platforms. While the CLARIOstar and NEPHELOstar readers are most frequently used for assay development, the PHERAstars are typically used for drug screening campaigns.

Miniaturizing HTRF assays

The above-mentioned BMG LABTECH readers helped Céline Legros, Project Manager of the Screening Assay Development Division at Servier, to downscale and miniaturise HTS screening assays. High-quality data were still delivered but with a significant saving in reagents and time.

Three examples are illustrated. First, the successful downscaling of a HTRF® assay from the 384- to the 1536-well format is discussed. On the PHERAstar FSX, assay miniaturization was combined with “on-the-fly” detection (only one flash per well) and the further dilution of reagents. This resulted in high-quality Z´ values and a significant reduction in reagent usage and time.

For TR-FRET- and HTRF-based detection, the TRF laser of PHERAstar FSX provides a significant speed advantage, allowing the measurement of a full 1536-well plate in only 36 seconds while still keeping a Z´ > 0.8, as shown for these cellular and biochemical HTRF assays.


Downscaling a fluorescence quenching assay

The second example discusses the downscaling from a 384 to a 1536- well plate format of a fluorescence quenching assay on the PHERAstar FSX. Despite miniaturisation, the quenching assay still delivered good Z' values.

Finally, a fast binding kinetic was measured on the CLARIOstar. In order to cope with the fast kinetic and the number of samples of this assay, a specific strategy that included the use of the built-in reagent injectors was designed.

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