Enzymatic Metallography for Ultrasensitive Biodetection

描述（由申请人提供）： 已经发现了一种新的方法，其中与针对感兴趣位点的抗体或生物靶向剂连接的酶从溶液中快速且高度选择性地沉积金属。灵敏度和分辨率都高于替代技术，足以在明场光学显微镜中可视化单基因拷贝并枚举低拷贝数靶标，例如未扩增的基因，而无需油浸。背景绑定几乎不存在。即使在大分子水平上，信号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。该方法的变体现在将针对具有不同要求的四种不同应用进行优化。对于免疫组织化学，该过程将被细化为最高的特异性和再现性，然后在广泛的双中心研究中与传统的有机色原进行比较验证。将在100例病例中使用10芯组织微阵列对20种常用免疫组织化学分析物进行染色。对于DNA和RNA原位杂交和Southern印迹，将追求增强以最大限度地提高灵敏度：通过掺入金颗粒加速酶促代谢，并将该方法与基于聚合物的信号放大方法相结合。然后将评价套细胞淋巴瘤中细胞周期蛋白D1 mRNA的原位杂交检测，以及淋巴瘤中克隆性B和T细胞基因重排的Southern印迹检测，在每种情况下与放射自显影和荧光法进行比较。酶金相学也将适用于电子显微镜标记。将对该方法进行优化，以获得最高的样本穿透率、信号均匀性以及对信号大小和形状的控制。在测试系统中筛选后，将在电子显微镜研究中对新试剂进行严格评估，以确定不同蛋白质在微孢子虫孢子发育中的分布和功能。

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.