Assay Development
Helps to develop assays in a high-throughput format for functional discoveries.
Assay Miniaturization
The multi-well plate assays are often developed with 96-well plates in the laboratories. The assays needs to be transferred to384- or 1536-well plate-compatible assays in our facility. The goal of assay development focuses on miniaturizing the assays for 1536-well plates and on the delivery of robust and well validated assays for HTS.
Assay Types
Protein-based biochemical assays - These screens use purified proteins, substrates and small compound inhibitors in buffered solutions to produce an optical (fluorescent, luminescent, etc.) readout that monitors enzymatic or binding activity using high-throughput plate readers. Small compound inhibitors are ranked by the signals, referring to targeted protein function, in this type of screen.
Advantage: As the first step for design of target-specific small molecules. Simple readouts result in tremendous throughput. Small volume reactions (5 ul) reduce reagent costs.
Disadvantage: Small compounds may have low water-solubility and low membrane permeability. The promiscuity and toxicity cannot be evaluated. Single protein inhibition may not cause desired phenotypic change in cells or tissues due to redundant pathways.
Cell-based assays - These screens can be macromolecular or small-molecule screens that utilize cells plated in 96 or 384 or 1536 well plates to produce visual phenotypic or signal changes in the cells. The two measurement types (although others exist) of a cell-based assay are:
Uniform well readouts - These include cell viability assays and luciferase-based reporter assays. These assays can provide simple readouts, resulting in tremendous throughput.
High-Content Imaging readouts - The assays are designed to probe changes in cellular phenotypes (i.e. foci formation screens, nuclear and cellular morphology, localization of proteins, etc). The assays employ specialized high-content imagers to produce high content pictures which can be used to measure phenotypic changes. These assays are content rich and can provide in-depth analysis of cell images. In addition, they can also be "multi-plexed" with multiple probes and DAPI for DNA staining to provide information on several changes to a cell phenotype simultaneously.
Advantage: Cell based assays produce phenotypic changes affecting pathways directly associated with disease states and the target protein does not have to be known. Small-molecule cell-based screens ensure compound solubility, membrane permeability, non-toxic and effectiveness at low therapeutically relevant concentrations.
Disadvantage: Lower throughput than protein-based assays, much more technically difficult and much longer duration (days/weeks vs. minutes). Target of inhibitors are not definitively known.
Detection
Signal-based detection: Through the advanced, HTS-compatible instruments, the HTS facility supports a wide variety of detection modalities for assay measurements including: absorbance, fluorescence, time-resolved fluorescence, fluorescence polarization, fluorescence resonance energy transfer (FRET), time-resolved FRET, and luminescence. Cell-based and chemical assays in experimental systems that have been used in prior models have included but are not limited to; recombinant proteins, membrane fractions, cellular isolates, cell lysates, and cell lines.
Image-based detection: Image-based detection will be provided by independent instruments.
Assay Validation
Assays are validated manually by the core facility and evaluated to determine the level of automation and instrumentation needed for HTS services. The assay will be validated with positive and negative controls and the robustness of the assay (Z-factor/Z’) will be determined to assure that the assay can be automated.
During validation of the automation, manual steps are replaced systematically with instruments to ensure accuracy and precision/fidelity of the assay as observed in the manual process. After determining the sequence of events and instrumentation for the assay, a robotic sequence is created to integrate these assay procedures into an automated fashion.
Hit confirmation
After hits are identified from a high-throughput screen, the hits are confirmed and evaluated using the following methods:
1) Re-testing: Compounds that were found active against the selected target are re-tested using the same assay conditions used during the HTS.
2) Dose response curve generation: Several compound concentrations are tested using the same assay, an IC50 or EC50 value is then generated.
Further selectivity and secondary screening will be performed to confirm the identified hits through additional services.