Correlative Enzymatic and Gold Probes

描述(申请人提供)：将开发一种新型免疫探针，包含酶和共价连接的1.4 nm、3 nm、5 nm或10 nm金标记物。它们将用于在细胞和组织水平上通过使用荧光或显色底物的荧光或明场光学显微镜对具有生物学意义的目标进行相关的显微染色，并使用单一标记程序在大分子水平上通过电子显微镜进行染色。通过酶法沉积荧光底物，新的探针将实现免疫荧光标记和3、5或10 nm金标记的组合，这一过程以前被金颗粒的强烈荧光猝灭特性所禁止。通过消除标记实验之间的加工对样品形态的影响，将提高两个互补数据集的相关性。此外，这些探针将促进多种标记，在这种标记中，两个或更多个目标在光或荧光水平上通过不同的颜色区分，在电子显微镜水平上通过不同尺寸的金颗粒来区分。将使用各种合成交联剂原理来制备探针，并将所得到的探针的每一组分的标记性能与使用斑点、细胞和组织对照玻片上的光学显微镜以及电子显微镜的等效单标记物进行仔细的比较。满足灵敏度、特异度和样本穿透性目标的探针将在两个研究应用中进行评估：(I)极管蛋白在小孢子体中的定位；(Ii)哺乳动物细胞核中前mRNA转导和剪接因子的结构和定位研究。 这项研究将为研究人员提供新的工具，在细胞和分子水平上结合生物系统中正常和疾病过程的数据，使人们能够获得关于这些过程如何工作的基本新知识，这将支持改进的诊断学和治疗学的发展。此外，预计新的探针将能够比单独使用的任何一种检测组件具有更高的检测灵敏度，这可能会通过改进的分子诊断程序，如DNA和RNA原位杂交和低丰度靶标的免疫组织化学，用于癌症或其他遗传异常的早期检测。

DESCRIPTION (provided by applicant): A new type of immunoprobe will be developed, containing both an enzyme and a covalently linked 1.4 nm, 3 nm, 5 nm or 10 nm gold label. These will be used for correlative microscopic staining of biologically significant targets at the cell and tissue level by fluorescence or brightfield light microscopy using either a fluorescent or chromogenic substrate, and at the macromolecular level by electron microscopy, using a single labeling procedure. By enzymatic deposition of a fluorescent substrate, the new probes will enable combined immunofluorescent labeling and 3, 5 or 10 nm gold labeling, a procedure previously prohibited by the strong fluorescence quenching properties of the gold particles. Correlation of the two complementary data sets will be improved by the elimination of the effects on specimen morphology of processing between labeling experiments. In addition, these probes will facilitate multiple labeling in which two or more targets are distinguished by different colors at the light or fluorescence level, and different sized gold particles at the electron microscopic level. A variety of synthetic cross-linking rationales will be used to prepare probes, and labeling performance of each component of the resulting probe will be carefully compared with the equivalent singly labeled conjugate using blots, light microscopy on cell and tissue control slides, and electron microscopy. Probes meeting targets for sensitivity, specificity and specimen penetration will be evaluated in two research applications: (i) localization of polar tube proteins in microsporida, and (ii) structural and localization studies on pre-mRNA transciption and splicing factors in the mammalian cell nucleus. This research will provide researchers with new tools for combining data on normal and disease processes in biological systems at the cellular and molecular level, enabling the acquisition of fundamental new knowledge of how these processes work that will support the development of improved dignostics and therapeutics. In addition, it is anticipated that the new probes will be capable of higher detection sensitivities than either detection component used alone, and this may be used for the earlier detection of cancer or other genetic abberations through improved molecular diagnostic procedures such as DNA and RNA in situ hybridization and immunohistochemistry of low abundance targets.