Enzymatic Metallography for Ultrasensitive Biodetection

描述(由申请人提供)： 已经发现了一种新的方法，其中连接到抗体或生物靶向剂的酶直接指向感兴趣的位置，从溶液中快速且高度选择性地沉积金属。与替代技术相比，灵敏度和分辨率都更高，足以在不浸油的情况下可视化单基因拷贝并计数低拷贝数目标，如Brightfield光学显微镜中的未扩增基因。背景绑定实际上是不存在的。即使在大分子水平上，信号的分辨率和清晰度也非常高，初步研究表明，该方法还为电子显微镜免疫标记提供了高细胞和组织穿透性。此方法的变体现在将针对具有不同要求的四种不同应用程序进行优化。对于免疫组织化学，该过程将被改进以获得最高的特异性和重复性，然后在一项广泛的双中心研究中与传统的有机色素相比较进行验证。20种常用的免疫组织化学分析将在一系列100例中进行染色，使用10个核心组织的迷迭光。对于DNA和RNA原位杂交和Southern blotting，将寻求增强以最大限度地提高灵敏度：酶促金属化将通过掺入金粒子来加速，该方法将与基于聚合物的信号放大方法相结合。然后评价其在套细胞淋巴瘤中细胞周期蛋白D1mRNA的原位杂交检测，以及在淋巴瘤中克隆B和T细胞基因重排的Southern Blot检测，并与放射自显影和化学发光法进行比较。酶金相法也将用于电子显微镜标记。该方法将进行优化，以提供最高的样品穿透性、信号一致性以及对信号大小和形状的控制。在测试系统中进行筛选后，新试剂将在电子显微镜研究中与传统的胶体金探针以及共价金簇和纳米颗粒探针进行关键评估，以表征不同蛋白质在微孢子虫孢子发育过程中的分布和功能。

DESCRIPTION (provided by applicant): A novel process has been discovered in which an enzyme linked to an antibody or biological targeting agent directed to a site of interest deposits metal rapidly and highly selectively from solution. Sensitivity and resolution are both higher then with alternative technologies, sufficient to visualize single gene copies and enumerate low copy number targets such as unamplified genes in the brightfield light microscope without oil immersion. Background binding was virtually non-existent. Signal resolution and clarity are very high even at the macromolecular level, and preliminary studies indicate that this method also provides high cell and tissue penetration for electron microscopy immunolabeling. Variations of this method will now be optimized for four different applications with different requirements. For immunohistochemistry, the process will be refined for highest specificity and reproducibility, then validated in comparison with conventional organic chromogens in an extensive two-center study. 20 commonly used immunohistochemical analytes will be stained in a series of 100 cases, using 10-core tissue midiarrays. For DNA and RNA in situ hybridization and Southern Blotting, enhancements will be pursued to maximize sensitivity: the enzymatic metallation will be accelerated by incorporation of a gold particle, and the method combined with polymer-based signal amplification methods. It will then be evaluated for the in situ hybridization detection of cyclin D1 mRNA in mantle cell lymphomas, and for the Southern Blot detection of clonal B and T cell gene rearrangements in lymphomas, in each case in comparison with autoradiographic and chemiluminescent methods. Enzymatic metallography will also be adapted for use in electron microscopy labeling. The method will be optimized to give highest specimen penetration, signal uniformity, and control over signal size and shape. Following screening in test systems, the new reagent will be critically evaluated against both conventional colloidal gold probes and against covalent gold cluster and nanoparticle probes in electron microscopic studies to characterize the distribution and function of different proteins in developing microsporidia spores.