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，它将为用户提供一种方法，在两个小时的程序中检测到低至0.001到0.0002毫摩尔(10-21到2×10-22摩尔)的蛋白质靶标。高灵敏度将通过两种方式提供。首先，新的探针将紧密结合酶标记和金纳米标记。当用金相衬底显影时，它们通过两个敏感且相辅相成的过程产生信号：金粒的自动金相显影和酶催化的金属沉积，称为酶金相。这种组合将提供高达100倍的灵敏度，比任何一种方法单独使用。其次，酶-金结构的多个拷贝，连同多个抗体或其他靶向试剂，将连接到一个可溶的聚合物骨架上，为探针提供内置的信号放大。新的偶联物将作为蛋白质印迹和其他蛋白质印迹方法的二次检测试剂。为了评估它们的性能，新的探针将在实验中与化学发光检测进行正面比较，以定量_1和_-4整合素和HER2蛋白的表达，并确定整合素和HER2蛋白表达的关系。在培养的细胞系中进行评估后，这些试剂将与化学发光法比较，以评估两系列乳腺癌病例中-1和-4整合素和HER2蛋白的表达，其中一种是HER2过表达的病例，另一种是EGFR过表达的病例。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HER2和整合素表达的相关性。然后将使用免疫沉淀和新试剂的Western blotting来研究_-4整合素与HER2蛋白的直接结合程度。该项目将开发一种新型的超灵敏检测试剂，它将在短时间内提高蛋白质印迹的常规检测灵敏度，并最终用于许多其他方法，如原位杂交和核酸印迹，这些方法的检测灵敏度至关重要。这将使得通过蛋白质印迹从较小的样本中更准确地定量蛋白质，以及更灵敏和精确地显示基因拷贝。这将使癌症、传染病和Pron疾病的诊断更快、更准确，改进治疗选择，并为分子水平的癌症研究提供新的工具。

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.