AMPLIFIED FLUORESCENT REPORTERS FOR AVIDIN-BIOTIN TESTS

拟议研究的长期目标包括制备亲和素、链亲和素和生物素衍生物，用扩增的荧光标签标记，用于各种亲和素生物素技术应用，包括免疫学、FISH诊断和流式细胞术。所提出的报告放大共轭物将增加信噪比，增强灵敏度，并提高检测分辨率。使用原型放大标签的初步结果显示了它们提供信号放大的潜力。本课题重点对标记结构进行修饰，进一步提高标记的适用性，具体目标如下：1)12个新型扩增荧光标记的合成与评价；2)标记与亲和素、修饰亲和素和链亲和素的偶联，3)标记蛋白及其生物素结合的荧光学研究，4)用荧光显微镜检测扩增蛋白偶联物与标准FISH分析中的商业试剂的比较，5)合成和评价扩增标记的生物素衍生物作为亲和素-生物素复合物的检测试剂。关于扩增共轭物结构要求的假设将通过与商业产品的直接比较来验证。该项目的健康相关性是基于在公认的诊断方案中应用扩增报告。未来的发展将专注于流式细胞术和共振能量转移研究的商业应用。拟议的商业应用：放大荧光探针在生物学研究和医学诊断方面具有相当大的商业潜力。

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.