NON-RADIOACTIVE SOUTHERN BLOTTING DETECTION SYSTEM

基于1.4 nm纳米金簇标记和自动金相增强，将开发快速、方便的检测和可视化试剂用于Southern blotting。在免疫斑点印迹中，具有自动同源显影增强的纳米金已经显示出与放射自显影和化学发光相媲美的检测灵敏度，并已显示出0.1pg(10[-18]摩尔)的目标免疫球蛋白；原位杂交，纳米金与醋酸银自体显影已被用于在石蜡包埋细胞和光镜活检切片中显示仅1-2个拷贝的目标病毒DNA。将测试三种方法：(A)检测带有次级纳金结合物的半抗原化杂交探针；(B)通过酶标记的杂交探针催化沉积纳米金标记的底物；(C)使用半抗原化探针催化报告沉积(CARD)，将纳米金-链霉亲和素作为最后一步。纳米金将通过对其配位配体的合成修饰来优化，以实现最小的非特异性结合和最高的可能灵敏度。将使用一种新的金基自动金相术系统，它可以产生更高的灵敏度和更低的本底，并开发专门的封闭剂来防止任何一种成分的非特异性膜结合。一套试剂包和程序将在几个小时内开发用于Southern杂交检测，而无需胶片曝光、暗室条件或放射性。拟议的商业应用：1998年，预计有近56.48万美国人死于癌症，每年用于癌症的成本估计为1070亿美元。Southern blotting是检测和表征癌症的一种主要方法：这些试剂将使Southern blotting更快、更简单地用于癌症诊断和研究，而不会有放射性探测器的危险和费用。它们的高空间分辨率也可能导致在高分辨率或高通量自动化系统中的应用。

Fast, convenient detection and visualization reagents will be developed for Southern blotting, based upon the 1.4 nm Nanogold gold cluster label combined with autometallographic enhancement. In immunodot blotting Nanogold with autometallographic enhancement has already exhibited detection sensitivities comparable with autoradiography and chemiluminescence, and has visualized 0.1 pg (10[-18] mol) of a target IgG; In in situ hybridization, Nanogold with silver acetate autometallography has been used to visualize as few as 1-2 copies of target viral DNA in paraffin-embedded cells and biopsy sections for light microscopy. Three approaches will be tested: (a) detection of a haptenated hybridization probe with a secondary Nanogold conjugate; (b) Catalytic deposition of Nanogold-labeled substrates by an enzyme-conjugated hybridization probe; and (c) Catalyzed Reporter Deposition (CARD) with Nanogold-streptavidin as the final step using a haptenated probe. Nanogold will optimized by synthetic modification of its coordinated ligands for minimal non-specific binding and highest possible sensitivity. A novel gold-based autometallographic system which produces higher sensitivities and lower backgrounds will be used, and specialized blocking reagents developed to prevent non-specific membrane binding of either component. A reagent package and protocols will be developed for Southern blot hybridization detection in a few hours without film exposure, darkroom conditions or radioactivity. PROPOSED COMMERCIAL APPLICATION: In 1998, nearly 564,800 Americans are expected to die of cancer, and the annual costs for cancer are estimated to be $107 billion. Southern blotting is a primary method for detection and characterization of cancer: these reagents will enable faster, simpler Southern blotting for cancer diagnosis and research without the hazard and expense of radioactive probes. Their high spatial resolution may also lead to applications in high-resolution or high-throughput automated systems.