Enzymatic Metallography for Biological Detection

描述了一种新的信号放大程序，其中金属而不是有机分子通过与抗体或其他生物靶向探针结合的酶催化沉积在标记位点。初步实验表明，新的系统是非常有效的原位杂交检测：检测灵敏度与automographically增强的纳米金探针与酪胺信号放大（TSA;也称为催化报告沉积，或CARD），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，并产生一个黑色的信号，很容易通过明场显微镜读取。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。将使用免疫印迹和免疫组织化学对照载玻片染色优化新工艺，以获得最高灵敏度和再现性，然后在组织切片中进行评价。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程序。对于原位杂交，新方法将与CARD-纳米金-自动荧光照相术进行比较，并与纳米金偶联酶底物直接沉积在感兴趣的位点的替代程序进行比较。拟议的商业应用：一种新的，普遍适用的检测系统将被开发作为辣根过氧化物酶/二氨基联苯胺（DAB）的方法，这是目前广泛使用的免疫组织化学染色的替代。与其他信号放大方法相比，新的过程更简单，可以实现相同的灵敏度;它产生一个密集的黑色信号，像DAB一样，很容易通过标准明场显微镜观察到。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，载玻片染色和原位杂交仪器。

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.