AMPLIFIED FLUORESCENT REPORTERS FOR AVIDIN-BIOTIN TESTS

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The long-term objectives of the proposed research include preparation of avidin, streptavidin, and biotin derivatives labeled with amplified fluorescent tags for use in a variety of avidin-biotin technology applications, including immunology, FISH diagnostics, and flow cytometry. The proposed reporter amplified conjugates will increase signal-to-noise ratio, enhance sensitivity, and improve resolution of detection. Preliminary results using prototype amplified tags demonstrated their potential to provide signal amplification. This research project focuses on modification of the labels' structures to further enhance their suitability and includes the following specific goals: I) synthesis and evaluation of 12 novel amplified fluorescent labels: 2) conjugation of the labels with avidin, modified avidin, and streptavidin, 3) fluorimetric study of labeled proteins and their biotin binding, 4) comparison of amplified protein conjugates to commercial reagents in standard FISH analysis with fluorescent microscopy detection, and 5) synthesis and evaluation of amplified-labeled biotin derivatives as detection reagents for avidin-biotin complexes. The hypothesis concerning structural requirements of the amplified conjugates will be validated by direct comparison to commercial products. The health-relatedness of the project is based on application of the amplified reporters in well accepted diagnostic protocols. Future development will address specifically commercial applications in flow cytometry and resonance energy transfer studies. PROPOSED COMMERCIAL APPLICATIONS: There is a considerable commercial potential for the amplified fluorescence probes in biological research and medical diagnostics.