The aim of an antibody incubation with a specimen, whether it be a section, cells in suspension, a tissue slice, a blotted membrane, or a rapid diagnostic test assay, is to achieve the most intense specific signal and the least non specific background possible. How to get the best from gold conjugates describes this in detail but the initial choice of gold conjugate is also important as discussed here.
Direct or indirect labeling (antibodies)
Gold conjugates may be used directly or indirectly to label antigens as indicated below. In the direct method the gold is conjugated to the primary antibody, whether monoclonal or polyclonal. This allows a single incubation to be performed and provides the simplest detection system. In the indirect method a primary unlabelled antibody is applied to the specimen to locate the antigen. This is followed by a gold labelled secondary that detects the primary antibody. This gold labelled secondary antibody is almost always an affinity purified polyclonal. In this way an amplification of the signal is achieved, often up to 10x compared with a direct incubation, according to the particle size.
The choice of the most appropriate system depends upon the type of component to be detected and what binding proteins are available. Some guidelines on making this choice are given below.
An extension of the indirect method may be used where there is no appropriate gold labelled secondary to match the primary antibody. For example if the unlabelled primary is from Pig and there is no matching gold labelled anti-Pig then an unlabelled anti- Pig (say Rabbit anti-Pig) may be used as a second step followed by a gold labelled third antibody (say Goat anti-Rabbit) that will detect the second antibody.
The indirect method is the most common for studying cells and tissues and avoids the need to label every primary antibody. It provides a universal method for detecting any primary antibody from the same primary species. ie all Rabbit antibodies may be detected by Goat anti-Rabbit gold conjugates, etc. The indirect method is longer than the direct method because of the need for two (or three) separate incubations but is the more widely used. The table below indicates the secondary gold labelled antibodies to be used with any chosen primary.
| Primary Antibody | Secondary Antibody(Gold Labelled) | Product Description |
| Rabbit IgG | Goat anti-Rabbit IgG (H+L) | GAR |
| Mouse IgG | Goat anti-Mouse IgG(H)(Human abs) Goat anti-Mouse IgG(H+L)(Human abs) Goat anti-Mouse IgG(Rat abs) |
GAM GMHL GMTA |
| Mouse IgM | Goat anti-Mouse IgM(mu chain specific) | GAMM |
| Mouse IgG or Mouse IgM (mixed or unknown) |
Goat anti Mouse IgG+IgM (H+L)(Human abs) | GAF |
| Mouse IgA | Goat anti-Mouse IgA | GAMA |
| Mouse IgG+IgM+IgA | Goat anti-Mouse IgG+IgM+IgA (polyvalent) | GAMP |
| Rat IgG | Goat anti-Rat IgG (H+L) (Human abs) Goat anti-Rat IgG(H+L) (Mouse abs) |
GAT GTMA |
| Human IgG | Goat anti-Human IgG(H+L)(gamma chain specific) Goat anti-Human IgG(H+L) |
GAH GAHL |
| Human IgM | Goat anti-Human IgM | GAHM |
| Guinea Pig IgG | Goat anti-Guinea Pig IgG (H+L) | GAG |
| Chicken IgG | Rabbit anti-Chicken IgG (H+L) | RCHL |
| Goat IgG | Rabbit anti-Goat IgG (H+L) Rabbit anti-Goat IgG (H+L)(Human abs) |
RAG RGHA |
| Sheep IgG | Donkey anti-Sheep IgG (H+L) | DAS |
Key:
(H+L) = heavy and light chain specific
(H) = heavy chain specific
(Human abs) = absorbed against human serum proteins
(Mouse abs) = absorbed against mouse serum proteins
(Rat abs) = absorbed against rat serum proteins
Absorption with serum proteins
Antibody-gold conjugates are absorbed against certain species of serum proteins as shown in the table above and as detailed in each Section. This absorption reduces cross reactions to those species to an absolute minimum.
When to use F(ab') fragments?
In some applications background labeling may be a problem due to the attraction of the Fc region of the antibody-gold conjugate to tissue components (called Fc receptors). Normally this is blocked by the simple application of normal serum prior to the first antibody (see How to get the best from gold conjugates). If the problem persists, however, then gold labelled F(ab') fragments of antibodies may be used. These conjugates are listed in each Section.
When to use antibodies, Protein A, Protein G or Protein A/G?
Protein A (PAG), Protein G (PGG) and Protein A/G (AG) are all separate proteins and will mimic a secondary antibody by binding to the Fc part of a primary antibody. In some situations they may be used in place of the secondary antibody. Unlike secondary antibodies, however, it is believed that there is only a single binding site for these proteins on the primary IgG. They do not bind with great affinity to IgM or IgA molecules.
The advantage of these proteins is that they provide a universal second step for a wide range of primary antibody species. They each bind with varying affinities to IgG molecules of different species as shown in the table below. However, if possible we would always recommend the selection of a species specific secondary antibody in preference to Protein A, G or A/G.
Relative affinities of specific Ig binding proteins
| Primary Antibody | Protein A: Old (PAG) | Protein G: Gold (PGG) | Protein A/G: Gold (AG) |
|---|---|---|---|
| Rabbit IgG | +++ | +++ | +++ |
| Mouse IgG IgM IgA |
++ +/- +/- |
++ +/- +/- |
++ +/- +/- |
| Rat IgG | +/- | + | +++ |
| Human IgG1 IgG2 IgG3 IgG4 IgA IgM |
+++ +++ +/- +++ + + |
+++ +++ +++ +++ - - |
+++ +++ +++ +++ - - |
| Guinea Pig IgG | ++ | ++ | ++ |
| Goat IgG | +/- | ++ | +++ |
| Bovine IgG | ++ | ++ | ++ |
| Sheep IgG | +/- | ++ | +++ |
| Chicken IgG | +/- | + | +/- |
Streptavidin or Goat anti-Biotin?
For the indirect detection of a biotinylated primary antibody or nucleic acid probe there is a choice of Streptavidin: gold or Goat anti-Biotin: gold conjugates. Historically Streptavidin has been the most frequently used detector for biotin because of the very high affinity constant between them. Most recently, however, Goat anti-Biotin has been shown to be a rather more sensitive detector of biotin compared to Streptavidin when conjugated to gold particles. This is because of the relatively large molecular size of the anti-biotin molecule (160,000 daltons) compared to Streptavidin (40,000 daltons) and the distance between the binding site of the gold on the Fc from the binding region of the antibody F(ab'). This is especially so when using larger gold particles but becomes insignificant for 5nm and 1nm gold conjugates. On occasions where background from non-specific attraction of Goat antibodies may cause a problem Streptavidin may provide a cleaner result. We recommend beginning with Goat anti-Biotin gold conjugates for the detection of biotin.
Cationic gold
Cationic gold allows highly sensitive and discrete microscopical studies of anionic (ie negative) sites in cells and tissues(1). The gold conjugate is made by careful conjugation to poly-L Lysine, a highly positive amino acid chain. A simple one step incubation of sections with the diluted Cationic Gold conjugate reveals subcellular sites having net negative charge. The charge distribution can be seen at a range of magnifications by using Cationic Gold of different particle sizes.
Most cells of eukaryotic origin have a net negative surface charge from anionic plasma membrane components. This charge distribution is thought to be important in movement of various soluble macromolecules across cell walls. Thus the role of the surface charge in cellular behaviour through interaction with neighbouring cells can be studied. For in vitro and in vivo studies Cationic Gold may also be used effectively to study the uptake of anionic material by endocytosis.
Reference (1): Bush MS and Allt G (1990) "Blood nerve barrier: distribution of anionic sites on the endothelial plasma membrane and basal lamina." Brain Res 535, 181 -188.
How to Choose the Gold Particle Size
A wide range of particle sizes can be conjugated to proteins and macromolecules. In principle smaller gold particles produce a higher labeling intensity on the specimen. This is because of the reduced steric hindrance to antigen detection. Typically an antibody of 160,000 daltons molecular weight will have a linear dimension of 8nm. Thus a 1nm gold particle, attached to the Fc region will hardly impede the antibody activity. A 20nm particle, however, while being more visible, will produce a greater steric hindrance by its proximity to the antigen binding region of the antibody. In addition the increased charge repulsion between larger particles reduces the number of labelled antibodies gaining access to the target antigen.
Different particle sizes are appropriate for different types of application as described below.
a) Light Microscopy (see Light Microscopy section)
Gold particles cannot easily be seen in the light microscope by bright field viewing. They must thus be silver enhanced for greatest visibility. Small gold particles will give the greatest number of gold labeling on the specimen and each particle can then be subsequently silver enhanced for maximum visibility of signal. A choice can be made between 1nm and 5nm gold conjugates. The 5nm conjugates are used for most standard purposes and are recommended for initial studies. For even greater labeling intensity the 1nm gold conjugates are preferred. In both cases the gold signal is silver enhanced to make it visible in the light microscope. The 1nm gold conjugates are particularly useful when penetration into cells and tissues (eg preembedding labeling) is required. Silver enhancing is described on page for our Silver Enhancement Kit
b) Transmission Electron Microscopy (see Gold Conjugates for EM)
For electron microscopy any particle size may be employed. For low magnification work larger particles (eg 15-30nm) are more easily seen. For high magnification studies the smaller particles (eg 5-10nm) are preferred. Again, smaller particles give higher labeling intensity and may be subsequently silver enhanced on the section to produce larger particles with this high labeling intensity. For ultra high sensitivity the 1nm gold particles can also be chosen but are usually visualised only after silver enhancing on the section.
For those just beginning immunogold labeling in the EM or performing studies over a range of magnifications, 10nm gold conjugates are recommended.
Scanning Electron Microscopy
The resolution of the scanning electron microscope indicates that larger gold particles (eg >20nm) should be used for detection by back scattering electron signals. However, as mentioned above, a greater intensity of labeling is achieved with smaller gold particles which can be subsequently silver enhanced with the LM/EM Silver Enhancing Kit SEKL 15. We would therefore recommend that a choice is made for SEM studies between 20 or 30nm gold conjugates for direct (unenhanced) viewing, or 5nm conjugates which can be simply silver enhanced within minutes for observation at low magnifications. With the latter approach each gold particle grows spherically to approximately 30nm in 3-5 minutes. The reaction is simply stopped by washing in water.
Blotting (see Gold Conjugates for Blotted Proteins)
For blotting applications where the gold conjugates are to be applied to proteins immobilised on nitrocellulose membranes, the choice is between 1nm, and 20nm gold conjugates. Even 5nm conjugates may also be used very effectively (LM Grade) together with subsequent silver enhancing. For ultra high sensitivity 1nm conjugates are preferred in combination with silver enhancing. However the 20nm gold conjugates allow blotted proteins to be identified on the immobilising membrane without silver enhancing. The 1nm and 5nm (LM Grade) gold conjugates, because of their much reduced steric hindrance and greater particle density in solution, may be diluted much further to provide the same final intensity compared with the 20nm conjugates. The 1nm and 5nm (LM) gold particles must be enhanced with the Blotting Silver Enhancing Kit.
Glycerol or Non Glycerol?
SPI-Mark standard Gold Conjugates are presented in a buffer containing 20% glycerol to allow good freezing characteristics. In most cases this causes no difficulties since the gold conjugates may be used highly diluted and specimens are fixed and embedded. On some occasions, however, when using the gold conjugate at high concentrations (eg 1:2) the presence of high glycerol content may interfere with satisfactory labeling of cells. Such specimens may include fixed cells in suspension or in cell culture where the glycerol may cause shrinkage of the cells. Such high concentrations of glycerol may also cause a lack of clarity of the gold particles in the EM and may even slow down the rate of labeling through increased viscosity. For such applications it is recommended that gold conjugates without glycerol are used (using the same product code number but identified as Non Glycerol - NG).
Technical Assistance
In addition to the choice of antibodies and particle size, the choice of the correct gold conjugate depends on the correct use of protocols for the specimen preparation and incubations. At SPI Supplies, we are ready to give assistance in these methods and will gladly help in the choosing of the correct gold conjugates for any specific application.