Gold colloidal particles

Fig 2 Immunogold negative staining, with a monoclonal antibody (JIM 5 (13)) that recognises a relatively unesterified pectic epitope, of rhamnogalacturonans extracted from onion cell walls. Arrows indicate 5 nm colloidal gold particles. Scale bar represents 200nm. 

One particularly novel carrier was reported to consist of 50-70 nm colloidal gold particles of the type often used in cytochemical labeling techniques for microscopy (Pow and Crook, 1993) (Chapter 24). Adsorption of peptide antigens onto gold and subsequent injection of the complex into rabbits in an adjuvant mixture resulted in rapid production of antibody of extremely high titer. The resultant antibodies could be used in immunocytochemistry at dilutions from l-in-250,000 down to l-in-1,000,000, which is orders-of-magnitude beyond the dilutions typically used with lower-titer antibodies. 

Reflux for 30 minutes. The color of the suspension will change from a dark blue to a red as the mono-disperse colloidal gold particles are formed. 

Figure 24.3 Antibodies coated on colloidal gold particles can be used to detect specific antigens in cells.

Pow, D.V., and Morris, J.F. (1991) Membrane routing during exocytosis and endocytosis in neuroendocrine neurons and endocrine cells Use of colloidal gold particles and immunocytochemical discrimination of membrane compartments. Cell Tissue Res. 264, 299-316. 

Fig. 1. Diagram of an EM immunogold assay localizing a protein on plastic sections. The primary antibody binds to an exposed surface epitope of the embedded cells. The antibody is then visualized by binding a second antibody coupled to a colloidal gold particle. The electron-dense gold particle visibly marks the position of the bound antibodies when visualized with the electron microscope. 

Geuze H, Slot J, Van Der Ley P, Scheffer R. Use of colloidal gold particles in doublelabeling immunoelectron microscopy of ultrathin frozen tissue sections. J Cell Biol 1981 89 653-665. 

Immunohistochemical labeling for electron microscopy is based on the same principles as immunohistochemistry for light microscopy. The differences are that specimen sections must be much thinner (50 100 nm) and the label must be electron-dense. The first electron-dense labels used for immunolabeling at the electron microscope level were ferritin and peroxidase. Peroxidase label can be visualized using DAB reaction product which becomes electron-dense after osmi-cation. With the advent of colloidal gold particles as markers in immunocytochemical... 

For a complete functional study of a biological pathway, it is often necessary to confirm the important protein interactions by in vivo experiments. This can be done by demonstrating protein localizations on a microscopic level, for instance, by tagging proteins with the green fluorescent protein or localizing them with antibodies and colloidal gold particles using an electron microscope. Additional, very specific biochemical experiments are often required to confirm the putative protein function. 

A report of a particle-based FPIA<56) details improvement in the limit of detection by two orders of magnitude over a conventional FPIA format for rabbit IgG, to 10 ° M. Goat anti-rabbit capture antibody (binder) is immobilized on polystyrene microspheres or colloidal gold particles, and the sample competes with a fluorescein-labeled rabbit IgG probe. The observed increase in sensitivity is apparently due to the large increase in effective mass of the binding complex. The polarization range is rather limited, though, reportedly due to low concentrations of immobilized antibodies. 

Other labels that have particular uses for electron microscopy are ferritin (11) and colloidal gold particles (12,13) (see Chapters 40-45). Gold particles are available in different sizes, therefore allowing simultaneous detection of several components on the same sample. Colloidal gold may also be detected with the light microscope following silver enhancement (see Chapter 29). In addition, radioactive labels have found some use in both light and electron... 

In another application, an optical trap was used to manipulate cell-surface proteins (29). First, colloidal gold particles conjugated to monoclonal antibod-... 

Muhlpfordt, J. A. (1982) The preparation of colloidal gold particles using tannic acid as an additional reducing agent. Experientia 38, 1127-1128. 

The lack of a clearly developed peak in the UV-visible absorption spectrum due to plasma resonance places a limit on the collective metallic behavior of the electrons in the cluster. On the other hand, the 5d -y 6s,6p interband absorption is well developed toward that of large colloidal gold particles. 

Annealing the particulate film (transferred to a quartz substrate) at 140 °C for 10 min led to the development of colloidal gold particles showing a characteristic plasmon absorption band with a maximum at 560 nm (Fig. 94) [110]. 

The modification to the Brust-Schiffrin method through the addition of lauryla-mine (LAM) or octadecylamine (ODA) instead of thiols to colloidal gold particles... 

It has been reported that some commercial preparations of colloidal gold-antibody complexes may contain free active antibody. Such free antibody will compete with antibody-colloidal gold particles for antigen binding sites and may reduce labeling intensity. The presence of free protein may be identified using a simple test procedure (20)... 

---