The Cytoblot: a Miniaturized High-Throughput Immunodetection Screening Assay Kit
Summary Background: A major goal of drug discovery efforts is to develop successful high throughput assays. Current assays either detect specific protein-ligand interactions using recombinant proteins or study the effects of small molecules on cell proliferation. The detection of cell proliferation has traditionally been performed by tedious and labor intensive methods, such as direct cell count, mitotic index, and clonogenic assays. Although these methods are useful, they are not practical for high-throughput screens, where a large number of compounds are assayed. Their low sensitivity also restricts usefulness in higher density formats where the number of metabolically active cells may be very low in comparison to the sample size. Moreover, many of these assays are employed without concern for the specific signaling pathway involved. As a consequence, there is a need to develop miniaturized cell-based assay suitable for high-throughput screening and applicable across a broad range of genetic-like screens in mammalian cells.Invention: A miniaturized high-throughput immunodetection screening assay (cytoblot) for discovery of lead compounds in drug development. The high density platform is composed of 6144 wells spaced only 1mM apart and utilizes DNA synthesis, gene expression, and mitosis markers as measures of a small molecules potential efficacy. In one example, the ability of cytoblotting to detect changes in DNA synthesis were measured by incorporation of 5-bromodeoxyuridine (BrdU, a thymidine analog) in the presence or absence of test compounds extracted from marine sponges. Mink lung cell were seeded in 6144 arrayed "nanowells" and treated with varying concentration of test compounds for 16 hours. Using an antibody raised specifically against the BrdU analog, the cytoblot assay was able to identify extracts that inhibited BrdU incorporation. The miniaturized format was also utilized for detection of post translational modifications of endogenous proteins, such as acetylation of histone H4 and phosphorylation of histone H3 and nucleolin. Importantly, as few as 500 cells in a 250 nl cell culture in a 6144-well plate could be detected with each of the respective antibodies to histone H4, histone H3, and nucleolin.
Applications Advantages: This method is a perfect complement to split pool synthesis methods for large-scale library production. The detection system is 10-fold more sensitive than enhanced chemiluminescence. The miniaturized format allows for reduced sample size (250 nl/well), cell number (500 cells/well), and reagent cost. One can also determine functional profiles for chemical compounds in multiple contexts, such as cells with different genetic backgrounds, tissue origins, and/or stages of development.
Commercial Applications: The cytoblot assay is utilitarian in nature and can be utilized for any number of potential screening markers in multiple contexts. These include cell proliferation (BrdU assay) and post-translational modifications (phosphorylation and acetylation markers) for testing of compounds that induce cell cycle arrest, inhibit signaling pathways, or induce apoptotic death.
Market: The market for the cytoblot assay is large and stable. This is due to nearly every disease state having a biological marker that indicates pathology. These disease states include diseases such as cancer, cardiovascular disease, and diabestes.
Publications: Brent R. Stockwell, Stephen J. Haggarty, and Stuart L. Schreiber. High-Throughput Screening of Small Molecules in Miniaturized Mammalian Cell-Based Assays Involving Post-Translational Modifications'' Chemistry AMPERSAND Biology 1999, 6, 71-83
Patent Status: Pending For Further Information Please Contact the Director of Business Development Laura Brass Email: [email protected] Telephone: (617) 495-3067
Inventor(s): Schreiber, Stuart L.
Type of Offer: Licensing
« More Pharmaceutical Patents