SBIR-STTR Award

Although cytokines are intimately involved with immune response and immunopathology, research eventually leading to the development of clinically relevant assays has been hindered by the sensitivity and versatility limitations of the ELISA assay. The aim of this proposal is to develop a flow cytometry based ultra-sensitive immunofluorescent assay system using capture by paramagnetic microparticles capable of measuring the level of cytokines in non-pathologic plasma or serum samples. The advantage of analysis by flow cytometry is the high degree of sensitivity afforded by tunable photomultiplier tubes enabling a movable 4-log range and the ability to correlate multiple analytes simultaneously. Preliminary paramagnetic microbead immunofluorescent assay systems have been developed for the simultaneous analysis of one, two, or three analytes based on antibody-cytokine-antibody-fluorochrome sandwiches and fluorochomecytokine competition assays having sensitivities of <50 pg/ml for human IL-2, IL-4, and TNF-alpha and can be completed in 1 hour. The focus of this proposal is to refine the assay by enhancing sensitivity and to develop further the capacity to analyze more analytes simultaneously. This study will lead to the development of a series of products intended for basic and clinical research market.Proposed commercial application:Development of paramagnetic microparticle based ultra-sensitive immunofluorescent assay capable of measuring multiple cytokines simultaneously by flow cytometry for the basic and clinical research market.National Cancer Institute (NCI)

Cytokines are intimately involved with immune response and immunopathology. Research leading to the development of clinically relevant assays has been hindered by the sensitivity and versatility limitations of the ELISA assay. The aim of this proposal is to develop a flow cytometry based ultra-sensitive solid phase immunofluorescent assay system for measuring the level of cytokines in non-pathologic and pathologic plasma, serum samples, or culture supernatants. Assays have been developed for IL-I alpha, IL-2, IL-4, TNF-alpha, and GM-CSF which can enable detection of less than 100 fg/ml and can be completed in l hour. The assay uses antibody-coated paramagnetic microparticles for analyte capture and a fluorescently labeled reporter antibody. Analyte concentration is proportional to fluorerescence. Different fluorochromes conjugated to the reporter antibodies and two different sized particles enabled simultaneous analysis of all 5 analytes. The focus of this proposal is to obtain optimal antibody pairs, define sensitivity levels and assay limitations, and to increase the number of different cytokine assays up to 15. This proposal will lead to the development of a series of products for Cytokine analysis and other analytes which can measure clinically relevant cytokine concentrations.Proposed commercial application:Development of microparticle based ultra-sensitive immunofluorescent assays capable of measuring multiple cytokines simultaneously by flow cytometry for the basic and clinical research market. These assays may be used for establishing normal ranges as well as pathologically associated changes, in cytokine levels and profiles, which in turn may result in clinical test kits.Thesaurus termscytokine, flow cytometry, immunoconjugate, immunofluorescence technique, method development biotechnology, biotin, chemical binding, fluorescein, magnetism, microcapsule, monoclonal antibody, phycobilinNational Cancer Institute (NCI)