Enzymatic Metallography for Biological Detection

A new signal amplification procedure is described in which metals, rather than organic molecules, are catalytically deposited at a labeling site by an enzyme bound to an antibody or other biologically targeted probe. Preliminary experiments suggest that the new system is highly effective for in situ hybridization detection: detection sensitivities were comparable with autometallographically enhanced Nanogold probes used with tyramide signal amplification (TSA; also known as catalyzed reporter deposition, or CARD), suggesting detection thresholds equal to or better than routinely used DAB staining methods. The new method combines high sensitivity and simplified protocols, and yields a black signal is easily read by brightfield microscopy. In addition, it gives high contrast and spatial resolution for electron microscopy immunolabeling. The new process will be optimized for highest sensitivity and reproducibility using immunoblots and immunohistochemical control slide staining, then evaluated in tissue sections. For immunohistochemistry, a series of antigens will be stained using the new method in parallel with routine immunohistochemical procedures using DAB on an automated stainer. For in situ hybridization, the new method will be compared with CARD-Nanogold-autometallography, and with an alternative procedure in which Nanogold-coupled enzyme substrates are deposited directly at the site of interest. PROPOSED COMMERCIAL APPLICATIONS: A new, universally applicable detection system will be developed as an alternative to the horseradish peroxidase/diaminobenzidine (DAB) method which is widely used now for immunohistochemical staining. Compared with other signal amplification methods, the new process is simpler and can achieve equal sensitivity; it generates a dense, black signal which, like DAB, is readily visualized by standard brightfield microscopy. The new detection method will be used in biomedical research for immunostaining and in situ hybridization. Many of these processes are now automated, and the new reagents will also be carefully optimized for use in automated tissue processing, slide staining and in situ hybridization instruments.