Over 50 years ago fluorescent labels were conjugated with antibodies for the first microscopical detection of antigens in tissues. In 1970 enzyme labels first provided a permanent colored stain with higher resolution and greater stability than fluorescent tags. With the advent of gold labels in 1971 sensitive high resolution immunocytochemistry became possible in the electron microscope for the first time. The subsequent development of silver enhancing methods then allowed gold labels to provide a highly specific and sensitive method for visualising antigens in light microscopy.
Since their introduction to microscopy, gold labels have also become recognized as very important tools for detection and quantitation of proteins, antigens, and nucleic acids when used with other techniques such as blotting, flow cytometry, hybridisation, and DNA fingerprint identification.
More recently a very important use for gold conjugates has emerged in their incorporation into rapid test immunoassays. In these techniques the unique red color of the accumulated gold label, when observed by lateral or transverse flow along a membrane on which an antigen is captured, or by observation of the red color intensity in solution, provides an extremely sensitive method for detecting sub nanogram quantities of proteins in solution.
Gold labels act as markers for molecules that are otherwise invisible by eye or through other detection systems. A colloidal gold conjugate consists of a suspension of gold particles coated with a selected protein or macromolecule (such as an antibody). The gold particles may be manufactured to any chosen size from 1-250nm. This gold probe detection system, when incubated with a specific target, such as in a tissue section, will reveal the target through the visibility of the gold particles themselves.
In the electron microscope the particles are visible without further treatment. In the light microscope, however, the gold particles are made visible through a short and simple silver enhancing procedure. For detection by eye, gold particles will also reveal immobilized protein on a solid phase such as a blotting membrane through the accumulated red color of the gold sol. Silver enhancement of this gold precipitate also gives further sensitivity of detection.
The conjugation of selected proteins to gold particles depends upon at least three physical phenomena:
Charge attraction of the negative gold particle to positively charged protein Hydrophobic absorption of the protein to the gold particle surface Binding of the gold to sulphur (dative binding) where this may exist within the structure of the macromolecule.
Gold may be conjugated to a wide variety of molecules including proteins (eg antibodies), polypeptides, carbohydrates, polymers, polysaccharides, enzymes and nucleic acids. The conditions under which a gold conjugate is manufactured drastically affect its performance and stability.
There are several advantages in choosing gold particles as markers for microscopic and non-microscopic identification of target molecules.
The choice of particle size makes it possible to study antigens microscopically over a wide range of magnifications and provides the opportunity for multiple labeling of antigens on tissue sections. In the electron microscope labeling with gold particles is completely unequivocal. The label cannot easily be mistaken for any other tissue structure and is either present or not present. The resolution of this labeling in both EM and LM is thus higher than with any other method.
While fluorescent labels and enzyme based colour reactions may fade with time and light exposure, gold particles do not disappear but give a permanent label. Because of the particulate nature of the gold label, quantitation in the electron microscope is possible, even for more than one antigen by multiple labeling with different particle sizes.
In the light microscope, the intense brown/black stain produced by silver enhancement of the gold signal has been shown to give a greater overall sensitivity and far greater resolution than other immunocytochemical methods and tissues can be counterstained with all the usual staining procedures.
Unlike some enzyme based detection systems, gold probes are essentially non toxic, safe and easy to use. The good stability of a well made gold conjugate gives the product a long shelf life whether frozen or stored at 4° C. For the staining of proteins immobilised onto membranes, gold probes have unsurpassed detection sensitivity and resolution. Finally, and perhaps surprisingly, gold probes are inexpensive in their application and their high sensitivity often allows valuable primary antibodies to be diluted significantly further than with other systems.
What Determines High Quality?
Several essential features must apply to gold probes if they are to be considered reliable high quality reagents for experimental and diagnostic research.
Antibodies and Proteins
To begin with the raw materials, all antibodies and proteins used for conjugation should be affinity purified and of the highest quality. They must possess very strong affinity for the specific antigen and have high avidity to withstand severe incubation and washing conditions. Cross reactivity must be the lowest possible. We use only the highest quality antibodies and proteins for the manufacture of the SPI-Mark reagents, a policy which has been rewarded with consistently reliable products. This also extends over to the SPI packaging and shipping methods, as this class of products is only shipped refrigerated and via a courier service, in order to ensure that nothing has happened, in transit, to degrate the integrity of the product before it gets into the hands of the final customer.
The development in our Laboratories of special techniques for producing pure gold and silver colloids with the lowest available coefficient of variation (CV) of particle size means that our gold conjugates are consistent from batch to batch and give the same high performance time after time.
Sensitivity and Stability
Sensitivity is all important in immunolabeling for detection of low levels of antigens in cells and tissues. In order for a gold probe to produce a strong specific signal together with a low background the conjugation of antibody to gold particle must be complete and stable under a variety of incubating conditions. The gold particle must have the minimum effect on the activity of the antibody to which it is conjugated but be strongly enough absorbed to the surface to remain stable for years. For long term storage it should be possible to freeze a gold conjugate without loss of the antibody activity. All standard SPI- Mark gold conjugates may be frozen and stored over long time intervals without deterioration.
For electron microscopy a very low level of clustering is important, especially if quantitation is to be performed or high quality micrographs are required. Clustering indicates a poor absorption of protein with bridging between gold particles. Heavily clustered conjugates have low stability and poor sensitivity. At the very least gold conjugates should have greater than 85% singlets and certainly no clusters greater than triplets. All SPI-Mark EM Grade gold conjugates are made to this high specification.
The strict quality assurance procedures used at all stages during the manufacture of SPI-Mark gold reagents ensure that all of these criteria are fully met and that our products have the highest possible specification and performance.