SBIR-STTR Award

Analysis of the cytokine network in normal immunoregulation and in disease processes has benefited from the development of the immunospot assay which can interrogate individual cells for secretion of specific immunoreactive antigens. In the ELISPOT version of this assay, cells are settled onto a solid phase surface of capture antibodies for several hours, and the resulting secreted-and-captured antigen is detected by the precipitated product of an enzyme conjugated to a detector antibody. The result is a colored spot at the site of the antigen-secreting cell. This assay has proven to be exquisitely sensitive, as it captures secreted protein immediately after release and prior to diffusion into a larger volume. The assay has also proven to be uniquely informative as it provides valuable information on cell to cell variation that is lost in ELISA assays run on secreted protein from pools of cells. The ELISPOT assay is currently used to characterize several normal immune functions and immune cell diseases, including leukemias, autoimmune conditions, and HIV/AIDS. A major limitation of the ELISPOT assay is that it only looks at one or, at most two, secreted products per cell. Trellis has previously developed a proprietary cell-based multiplex microscopy technology and has now extended it to the ELISPOT resulting in a true profiling immunospot assay, the CelISPOT. The Specific Aims of this Phase I proposal are to: 1. automate the present manual system for reading and classifying CelISPOT cytokine secretion patterns; 2. extend the assay from the current 5-plex murine system to encompass 9 human cytokines; and 3. use the system to classify human T cells according to the conventional Th1 and Th2 phenotypes, and determine the extent to which individual cells deviate from these classes. The overall result of this Phase I effort will be a fully functional platform for a multiplex immuno spot analysis. In Phase II of this project, we will extend the reagent set to 25 cytokines, and together with our external collaborators, we will compile a database of results in major disease indications to support commercialization in Phase IIl. The availability of multiplex immunospot analysis for a comprehensive complement of cytokines, with sufficient sensitivity to detect rare cells, is expected to have far-reaching application in clinical medicine. The CelISPOT assay's unique ability to characterize pathological phenotypes in detail will provide unprecedented precision and sensitivity in characterization of immune system function for a wide range of important diseases.

Thesaurus Terms:
cytokine, immunologic assay /test, method development enzyme linked immunosorbent assay, helper T lymphocyte, immunoregulation, phenotype

Immune cells use signals to coordinate their attack on an infectious agent or to downgrade their attack after danger has past. The signals that they use are proteins called cytokines, of which there are about 25. During immunological disorders, cytokines can either signal or control a self-directed attack on the body, and therefore much attention has been paid to cytokines during autoimmune conditions. Yet there are few ways to query individual cells for cytokine expression and until now there have been no ways to do so on the small samples of immune cells isolated from skin and other inflamed tissues. Trellis' approach to measuring single cell cytokine expression is to capture the cytokines produced by single cells while they lie on a flat plastic surface. These cell "footprints" can then be probed by multihued nanoscale beads, each bead type linked to a different anti-cytokine antibody. This method has already revealed that the language of immune cells is not as simple as it is sometimes portrayed. The high level of diversity of immune cells in normal samples may be much diminished in disease or cell types that are rare in normal tissue may become significantly amplified. This is one question that can be explored with Trellis' CellSpot-CK technology. Other questions relate to staging disease based on direct exploration of the cellular profiles that underlie disease manifestation. Such information should be very useful in selecting treatment regimens for these chronic, progressive diseases as well as for monitoring patient responses. Before introducing CellSpot-CK to cellular immunologists throughout the world, Trellis will need to make the instrument used to read CellSpots more robust, easier to use, and faster. This is one Specific Aim in this Phase II proposal. The other Specific Aims relate to characterizing the cytokine language of immune cells in both health and disease. The successful development of CellSpot-CK will make available to researchers a new tool for understanding the role of the immune system in many chronic conditions like eczema, psoriasis, arthritis, and asthma. It will also provide a tool to learn how immune cells respond to drugs under development as cures for these diseases. Longer term, it may provide an important clinical "theranostic" assay (therapy selection based on a diagnostic procedure).

Thesaurus Terms:
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