Enzymatic Metallography for Ultrasensitive Biodetection

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.