IN Cell User's Day 2009 講演要旨
Assay Development Considerations for High Content Screening; Hepatotoxicity and other key Drug Discovery Assays
Andrew Ball Ph.D, HCS Group Leader
Using a GE IN Cell Analyzer 1000 platform as a primary development tool, we have developed a large number of extensively validated High Content Screening assays for multiple stages in the drug discovery and development, from primary screening thru in vitro toxicity assessment. The quality of HCS data is directly related to the quality of the detection reagents employed. Antibodies for HCS carry special requirements. These include strong antigen affinity, minimal non-specific binding, minimized interactions between primary or secondary antibodies, and a sufficient signal:background ratio to ensure an adequate screening window. Additionally, to enable scale up of HCS assays, the assay protocol must be highly reproducible, and the reagents must exhibit minimal assay-to-assay variability. To date, we have successfully developed assays targeting hepatotoxicity, neurotoxicity, cell cycle control, cell signaling, and many cellular stress pathways. We will present data showing that it is possible to use HCS to characterize multiple cellular events in intact cells. For example, Millipore's Hepatotoxicity HCS assay comprises detection reagents and protocols for profiling up to eleven endpoints within a single assay - Cell Loss, Cell Cycle Arrest, DNA Degradation/Apoptosis, Nuclear Size, Oxidative Stress, Stress Kinase Activation, DNA Damage, Mitochondrial Membrane Potential, Mitochondrial Mass, Mitotic Arrest and Cytoskeletal Integrity. Our data demonstrate the assay’s effectiveness in the detection of cellular responses to toxins. The combination of unique antibody pairs and detection reagents allows comprehensive assessment and analysis of the cellular systemic response to toxin challenge. Another example includes data from our Neurotoxicity assays which demonstrate that by multiplexing measurements of neuronal morphology with cell count, HCS represents a more sensitive tool for analysis of neurotoxicity than traditional, widely used cytotoxicity assays. We will also present data showing the utility of our HCS assays for screening key drug targets like p38 MAPK, ERK and many others. In addition to serving as a screening tool, HCS has enabled high levels of quality control for each assay we have developed. Examples of this include extensive characterization of antibody performance, detection of lot-to-lot variation of reagents, assay validation in multiple cell types, and reagent stability. This type of validation data is critical to minimize inter- and intra- assay variation. In conclusion, Millipore has developed a large portfolio of highly-validated HCS assays representing comprehensive workflow solutions for drug screening, and for in vitro toxicology assessment - representing broad potential for drug discovery and development.
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