Polymeric Enzyme-Gold Probes for Ultrasensitive Protein Blotting

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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.