Polymeric Enzyme-Gold Probes for Ultrasensitive Protein Blotting

描述（由申请人提供）：我们建议开发用于蛋白质印迹的超灵敏检测试剂，其将为用户提供在两小时程序中检测低至0.001至0.0002阿莫尔（10-21至2 x 10-22 mol）的蛋白质靶的方法。将以两种方式提供高灵敏度。首先，新的探针将同时包含酶和金纳米颗粒标签。当用金相基质显影时，它们通过两个敏感且相互增强的过程产生信号：金颗粒的自显影和酶的催化金属沉积，称为酶金相。这种组合将提供比单独使用任何一种方法高100倍的灵敏度。其次，酶-金构建体的多个拷贝与多种抗体或其他靶向剂一起将缀合至可溶性聚合物主链，从而为探针提供内置信号放大。新的结合物将被用作Western印迹和其他蛋白质印迹方法中的二级检测试剂。为了评估它们的性能，新的探针将在实验中与荧光检测进行头对头比较，以定量整合素1和整合素4以及HER 2蛋白的表达，并确定乳腺癌中整合素和HER 2蛋白表达之间的关系。在培养的细胞系中进行评价后，将试剂与化学发光进行比较，以评估两个系列乳腺癌病例中的_-1和_-4整联蛋白和HER 2蛋白，一个是用HER 2过表达病例构建的，另一个是用EGFR过表达病例构建的。将免疫印迹结果与免疫组织化学染色、原位杂交和DNA微阵列数据进行比较，以评估HER 2和整合素表达之间的相关性。然后使用免疫沉淀法，然后用新试剂进行蛋白质印迹法，研究_-4整合素和HER 2蛋白之间的直接结合相互作用的程度。该项目将开发一种新型的超灵敏检测试剂，该试剂将在短实验时间内提高蛋白质印迹的常规检测灵敏度，并最终用于检测灵敏度至关重要的许多其他方法，如原位杂交和核酸印迹。这将使蛋白质印迹法能够更精确地定量来自较小样品的蛋白质，以及更灵敏和精确地可视化基因拷贝。这将能够更快、更准确地诊断癌症、传染病和朊病毒疾病，改善治疗选择，并为在分子水平上研究癌症提供新的工具。

DESCRIPTION (provided by applicant): We propose to develop ultrasensitive detection reagents for protein blotting which will provide users with a means to detect as little as 0.001 to 0.0002 amol (10-21 to 2 x 10-22 mol) of a protein target in a two hour procedure. The high sensitivity will be provided in two ways. Firstly, the new probes will incorporate both enzymatic and gold nanoparticle labels in close proximity. When developed with a metallographic substrate, they generate a signal through two sensitive and mutually reinforcing processes: autometallographic development of the gold particle, and catalytic metal deposition by the enzyme, known as enzyme metallography. This combination will provide up to 100 times greater sensitivity than either method alone. Secondly, multiple copies of the enzyme-gold construct, together with multiple antibodies or other targeting agents, will be conjugated to a soluble polymer backbone, providing the probe with built-in signal amplification. The new conjugates will be used as a secondary detection reagents in Western blotting and other protein blotting methods. To evaluate their performance, the new probes will be compared head-to-head with chemiluminescent detection in experiments to quantitate the expression of _-1 and _-4 integrins and HER2 proteins, and to determine the relationship between integrin and HER2 protein expression in breast cancer. After evaluation in cultured cell lines, the reagents will be compared with chemiluminescence for the assessment of _-1 and _-4 integrins and HER2 protein in two series of breast cancer cases, one constructed with HER2-overexpressing cases and one with EGFR-overexpressing cases. Comparison of the western blot results with immunohistochemical staining, in situ hybridization, and DNA microarray data will be used to assess the correlation between HER2 and integrin expression. The extent of direct binding interaction between _-4 integrins and HER2 protein will then be investigated using immunoprecipitation followed by western blotting with the new reagents. This project will develop a new type of ultrasensitive detection reagent that will increase routine detection sensitivity within a short experimental time for protein blotting, and ultimately for many other methods such as in situ hybridization and nucleic acid blotting where detection sensitivity is critical. This will enable more precise quantitation of proteins from smaller samples by Western blotting, and more sensitive and precise visualization of gene copies. This will enable faster, more accurate diagnosis of cancer, infectious diseases and prion diseases, improve therapy selection, and provide new tools for the study of cancer at the molecular level.