Enzymatic Metallography for Biological Detection
用于生物检测的酶金相学
基本信息
- 批准号:6403845
- 负责人:
- 金额:$ 10.6万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2001
- 资助国家:美国
- 起止时间:2001-09-30 至 2003-03-31
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
A new signal amplification procedure is described in which metals, rather than organic molecules, are catalytically deposited at a labeling site by an enzyme bound to an antibody or other biologically targeted probe. Preliminary experiments suggest that the new system is highly effective for in situ hybridization detection: detection sensitivities were comparable with autometallographically enhanced Nanogold probes used with tyramide signal amplification (TSA; also known as catalyzed reporter deposition, or CARD), suggesting detection thresholds equal to or better than routinely used DAB staining methods. The new method combines high sensitivity and simplified protocols, and yields a black signal is easily read by brightfield microscopy. In addition, it gives high contrast and spatial resolution for electron microscopy immunolabeling. The new process will be optimized for highest sensitivity and reproducibility using immunoblots and immunohistochemical control slide staining, then evaluated in tissue sections. For immunohistochemistry, a series of antigens will be stained using the new method in parallel with routine immunohistochemical procedures using DAB on an automated stainer. For in situ hybridization, the new method will be compared with CARD-Nanogold-autometallography, and with an alternative procedure in which Nanogold-coupled enzyme substrates are deposited directly at the site of interest. PROPOSED COMMERCIAL APPLICATIONS: A new, universally applicable detection system will be developed as an alternative to the horseradish peroxidase/diaminobenzidine (DAB) method which is widely used now for immunohistochemical staining. Compared with other signal amplification methods, the new process is simpler and can achieve equal sensitivity; it generates a dense, black signal which, like DAB, is readily visualized by standard brightfield microscopy. The new detection method will be used in biomedical research for immunostaining and in situ hybridization. Many of these processes are now automated, and the new reagents will also be carefully optimized for use in automated tissue processing, slide staining and in situ hybridization instruments.
描述了一种新的信号放大程序,其中金属,而不是有机分子,被酶结合到抗体或其他生物靶向探针催化沉积在标记位点。初步实验表明,新系统对原位杂交检测非常有效:检测灵敏度与使用酰胺信号放大(TSA,也称为催化报告沉积,CARD)的自动金相增强纳米金探针相当,表明检测阈值等于或优于常规使用的DAB染色方法。新方法结合了高灵敏度和简化的协议,产生的黑色信号很容易被明场显微镜读取。此外,它为电子显微镜免疫标记提供了高对比度和空间分辨率。新工艺将通过免疫印迹和免疫组织化学对照玻片染色进行优化,以获得最高的灵敏度和可重复性,然后在组织切片中进行评估。对于免疫组织化学,将使用新方法对一系列抗原进行染色,同时在自动染色机上使用DAB进行常规免疫组织化学染色。对于原位杂交,新方法将与card -纳米金-自动金相法进行比较,并与纳米金偶联酶底物直接沉积在感兴趣的位点的替代方法进行比较。建议的商业应用:将开发一种新的,普遍适用的检测系统,以替代目前广泛用于免疫组织化学染色的辣根过氧化物酶/二氨基联苯胺(DAB)方法。与其他信号放大方法相比,新方法简单,可实现等灵敏度;它会产生一个密集的黑色信号,就像DAB一样,很容易通过标准的明场显微镜观察到。新的检测方法将用于生物医学研究的免疫染色和原位杂交。许多这些过程现在是自动化的,新的试剂也将被仔细优化,用于自动化组织处理,玻片染色和原位杂交仪器。
项目成果
期刊论文数量(3)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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RICHARD DENIS POWELL其他文献
RICHARD DENIS POWELL的其他文献
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{{ truncateString('RICHARD DENIS POWELL', 18)}}的其他基金
Smaller, Brighter Probes for Correlative Super-resolution and Electron Microscopy
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Conductive Metallography for Serial Section Electron Microscopy at Nanometer Resolution
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8834483 - 财政年份:2015
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$ 10.6万 - 项目类别:
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Serial Blockface SEM Labels for Assessing Nervous System Plasticity
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Correlative Chromogenic Gene and Protein assessment
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$ 10.6万 - 项目类别:
Enzymatic Metallography for Ultrasensitive Biodetection
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- 批准号:
6883438 - 财政年份:2001
- 资助金额:
$ 10.6万 - 项目类别:
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