SPI-Mark™ Colloidal Gold Reagents
Introduction to Gold Labelling
Introduction to Gold Labelling
History
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 Probes
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.
Conjugation
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.
Why Gold?
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 labelling of
antigens on tissue sections.
In the electron microscope labelling 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 labelling 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 labelling 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.
Size distribution of SPI-Mark gold conjugates
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.
Gold Colloids
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 immunolabelling 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.
Clustering
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.
Quality Assurance
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.
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Friday July 04, 2008
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