The state-of-the-art in immunogold
technology is the result of the relentless efforts of many researchers
worldwide, who recognized the need for an unambiguous marker in electron
microscopy immuno detection. Drawing from their achievements, Aurion has
further developed this technology and the outcome is its proprietary products.
Aurion wishes to pay homage to all those who have contributed significantly to
the development of immunogold silver detection, and especially to those who
have supported Aurion during the years in its efforts to establish itself as a
partner in research and business. The following review is necessarily
The first publication using colloidal gold particles as markers in immunocytochemistry dates back to 1971 (Faulk & Taylor). Since then, there has been a steady increase in the number of publications. Initially and partly before the Faulk & Taylor publication the advances in this area were centered around the development of protocols for the production of discrete sizes of gold particles with acceptable variability (Faraday, 1857; Zsigmondy, 1925; Frens, 1973; Muhlpfordt, 1982). Conjugates with more homogeneous gold particle size could be obtained by gradient centrifugation. This was demonstrated in a milestone publication by Slot et al. (1981) who perfected protocols for the preparation and application of protein-A gold conjugates (first introduced by Romano & Romano in 1977). Among researchers who investigated the interaction and physico-chemical aspects of immunogold conjugate preparation the names of Geoghegan and Horisberger also deserve a prominent place. Further work by Slot and his colleagues brought out another important publication (Slot et al. 1985), introducing procedures to produce homogeneous particles and conjugates without gradient centrifugation. The homogeneity of the particles was so good that double and even triple labeling of multiple antigens finally became readily feasible. In retrospect, protein-A gold conjugates should rightfully be considered the classic immuno detection reagents for electron microscopy.
In the mid 80’s, the popular colloidal gold particle sizes were between 5 and 40 nm. However, as the homogeneity of the particle size was being perfected, a general consensus began to emerge that the labeling density is inversely related to the particle size. This realization ultimately became an important justification for pursuing the development of smaller gold particles.
Although the colloidal gold detection system was initially conceived as a marker system limited to electron microscopy, a.o. De Mey, Holgate, Springall and Scopsi recognized and demonstrated its value as histochemical marker in light microscopy. This application became possible, especially with the availability of a silver enhancement method introduced by Liesegang (1911) and further developed by the in-depth studies of Gallyas and Danscher. The enhancement protocol published by Danscher (1981) eventually provided researchers with a means of silver enhancement for both light and electron microscopy in a controlled manner. At about the same time immunogold conjugates and silver enhancement also found applications in immunoblotting (Brada, Moeremans 1984), thus, finally demonstrating their capacity as universally applicable detection reagents.
Around 1985, Leunissen, founder of Aurion, started his research on the further reduction of the particle size in colloidal gold conjugates at the electron microscopy facility of the Department for Molecular Cell Biology in Utrecht, The Netherlands. This provided him with the experience and the knowledge for the development of ultra small colloidal gold particles. The development led to the recognition that not only labeling density but also sensitivity and penetration of gold reagents are inversely related to gold particle size.
Since the founding of Aurion, Leunissen has focused his research on the further development of ultra small gold particle based detection and enhancement and on the concept of immunogold silver detection as an integrated system. He has developed a wide range of ultra small gold conjugates with subnanometer particle size, the unique incubation solution additive BSA-c™, silver enhancement reagents for light microscopy (R-Gent SE-LM) and more recently (1999) the first silver enhancement solution intended specifically for electron microscopy (R-Gent SE-EM).