2012 © Institute for Molecular Medicine Finland FIMM
Assay Development
Assay Development and HTS optimization
Assay development starts by identifying the target and its activity. The next step is determining whether the objective is to inhibit or activate the target. A great starting point for assay development is a bench scale assay for the target used by the principal investigator, who initiates the small molecule screen by contacting the FIMM Chemical Biology unit. The assay developers can also begin by reaching literature and set up the assay from scratch. A small molecule chemical screen can be cell-based or based on a biochemical assay. The assay developers at FIMM Chemical Biology unit set up the assay and optimize it for high throughput screening.
With positive and negative controls, we confirm that assay repeatedly gives a Z’-factor >0.5. The positive control can be either a small molecule inhibitor that inhibits the reaction in question or a reaction without the enzyme of interest. Next, we test DMSO tolerance since our compound libraries are dissolved in DMSO. For biochemical assays, a final DMSO concentration between 1 and 5% is often convenient – it allows for flexibility with compound addition and supports solubility of compound. Whole cell assays normally tolerate no more than 1% DMSO.
Besides the primary assay, we also preferentially develop one or more counter screens (similar assay where an inhibitor should not hit) and an orthogonal assay (assay designed to test for inhibition of the same process but with a different readout to sort out compounds interfering with the readout).
After primary assay development, the assay is miniaturized using HTS equipment to 384- or 1536-well format and re-tuned for high-density format. Finally, two full Z’-plates with negative and positive controls are prepared and measured to test the reproducibility and precision of the assay and to detect any positional plate effects and systematic pipetting errors.
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| Jani Saarela Senior Researcher, Assay Developer jani.saarela@fimm.fi +358 50 317 5485 |
Evgeny Kulesskiy Assay Developer evgeny.kulesskiy@fimm.fi +358 50 318 5140 |