Simultaneous detection

Multiple labelling using fluorescent labelled secondary antibodies.

Montage of six 20X images of triple-stained artery in human skin acquired on a CARV non-laser confocal microscope.

Simultaneous detection of more than one antigen depends on at least two important criteria:
1  Availability of secondary antibodies that
•  are derived from the same host species so that they do not recognise   one another
•  do not recognise other primary antibodies used in the assay system
•  do not recognise immunoglobulins, from other species possibly   present in the assay system
•  do not cross-react with the tissues or cells under investigation.
2  Use of probes (enzyme-reaction products, fluorophores, or
 electron-dense particles) that are well resolved.
It is obvious that care has to be taken when planning a multiple labelling experiment. Before starting any experiment, ask the following questions;
1 Are there potentially cross reactive species?
2 Are there any Fc receptors present?
3 Is there any endogenous IgG in the system?
4 Are the primaries of the same species?
5 Does the blocking reagent contain IgG?
The aim of each multiple labelling experiment is to ensure that each secondary antibody only binds to its respective primary and all other sites, immunoglobulins and other primaries must be blocked to ensure minimal cross reactivity.
For a review of multicolour immunofluorescence labelling with confocal microscopy see Brelje, Wessendorf, and Sorenson. “Multicolour laser scanning confocal immunofluorescence microscopy: Practical application and limitations.” In “Cell Biological Applications of Confocal Microscopy”. B. Matsumoto, ed. in Methods in Cell Biology. 1993. Academic Press, Inc., Orlando, Florida.

Whole molecules or F(ab’)2 fragments (Figure 1) are used when binding of whole molecule, secondary antibodies to Fc receptors on cell surfaces needs to be avoided. As an alternative, binding of whole molecule, secondary antibodies to Fc receptors may be blocked by incubating cells at 4°C in a buffer containing sodium azide and normal serum from the host species of the labelled secondary antibody. Please note that if a primary antibody is not an F(ab’)2 fragment, it may also bind to Fc receptors, and blocking with normal serum from the host species of the secondary antibody may not always be successful. Caution: Never block with normal serum or IgG from the host species of the primary antibody when using a labelled, secondary antibody.

According to Jackson ImmunoResearch testing, there appears to be no species-specific difference in the quality of antibody conjugates. Therefore selection of the host species should be based on other criteria.
For example, when using Protein A-agarose, to separate antigen-antibody complexes from other components, rabbit antibodies should be used since goat IgG, as well as IgG from other species, may not bind as well to Protein A. On the other hand, Protein G binds well to IgG from goat, sheep, donkey and rabbit.
Caution: When selecting a secondary antibody, avoid the use of antibodies that have been adsorbed against closely related species, unless it is absolutely necessary to detect one species in the presence of the other. Such antibodies may not react well with all subclasses of IgG, especially those subclasses which are most closely homologous to the species they were adsorbed against.
For example, do not use an anti-mouse IgG that has been adsorbed against rat IgG unless you are trying to detect a mouse primary antibody in rat tissue that contains rat immunoglobulin, or in some other tissue in the presence of a rat primary antibody. Conversely, if you wish to detect a mouse primary antibody in the absence of rat immunoglobulins, it is best to use an anti-mouse secondary antibody that has not been adsorbed against rat.

Human brain progenitor, cultured cells were immunocytochemically stained for GFAP (blue AMCA), Doublecortin (red Cy2), and Nestin (green RRX).

Caution: Antibodies against one species may cross-react with a number of other species, unless they have been specifically adsorbed.
Warning: Bovine serum albumin (BSA) and dry milk may contain IgG which reacts with anti-bovine IgG, anti-goat IgG, anti-horse IgG, and anti-sheep IgG antibodies. Therefore use of BSA and/or dry milk to block or dilute these antibodies may significantly increase background staining and reduce antibody titre.immunoglobulins share the same light chains (either kappa or lambda).
Anti-IgG, Fc fragment specific antibodies react with the Fc portion of the IgG heavy chain, and can be produced by adsorption against F(ab’)2 fragments. In some cases, such antibodies are additionally adsorbed to minimise possible cross-reactivity to IgM and/or IgA. In such cases (anti-human, anti-mouse, and anti-rat), they are referred to as gamma chain specific.
Caution: Anti-IgG (Fc) antibodies do not react to all subclasses of IgG as equally as either Anti-IgG (H+L) antibodies, or Anti-IgG F(ab’)2 fragment-specific antibodies. To avoid this potential problem Jackson ImmunoResearch make a goat anti-mouse IgG Fc gamma chain specific antibody with balanced reactivity with all four subclasses (1, 2a, 2b, and 3) of mouse IgG.
Anti-IgG, F(ab’)2 fragment specific antibodies are produced by adsorbing against Fc fragments and therefore react only with the Fab portion of IgG. Since they react with light chains, they also react with other immunoglobulins sharing the same light chains.

Minimised cross reactivity antibodies will have been tested by ELISA and/or adsorbed against the IgG and serum proteins of other species. They are recommended when the possible presence of immunoglobulins from other species may lead to interfering cross-reactivities. However, caution should be exercised when considering antibodies that have been adsorbed against closely related species since they have greatly reduced epitope recognition and may recognise some monoclonal antibodies very weakly.
Two examples of antibodies, which have diminished epitope recognition after adsorption with closely related species, are Anti-Mouse IgG (min X Rat and other species) and Anti-Rat IgG (min X mouse IgG and other species). Most multiple-labelling experiments require the use of minimised cross reactivity antibodies to minimise cross-reactivities to other species.

Fab fragments of affinity-purified, secondary antibodies are used to sterically cover the surface of immunoglobulins for double labelling primary antibodies from the same host species, or to block indigenous immunoglobulins on cell or tissue sections.
Monovalent Fab fragments of secondary antibodies may be used for these purposes for the following reasons. Whole IgG molecules and F(ab’)2 fragments of IgG have two antigen binding sites. After binding to its primary antibody (for example, goat anti-mouse IgG binding to the first mouse primary antibody), most of the secondary antibodies will still have one open binding site, which can capture the second primary antibody from the same species (for example a second mouse IgG primary antibody). Consequently, overlapping labelling of the two antigens will occur.
For selected literature references see Wessel and McClay, J. Histochem. Cytochem. 1986. 34, 703; Franzusoff et al., J. Cell Biology. 1991. 112, 27; Lewis Carl et al., J. Histochem. Cytochem. 1993. 41, 1273; and Negoescu et al., J. Histochem. Cytochem. 1994. 42, 433.
Monovalent Fab secondary antibodies should not be used when primary antibodies from the same host species are different classes of immunoglobulins, such as IgG and IgM.
It is also preferable not to use these when primary antibodies from the same host species are different subclasses of IgG, such as Mouse IgG1 and Mouse IgG2a. In these cases, class-specific or subclass-specific antibodies may be used to distinguish between the two primary antibodies.
It is also important to keep in mind that these antibodies have not been adsorbed to remove cross-reactivities to other species, and they might contribute to some degree of background staining for certain applications using the example below.
The following examples show two possible protocols that can be use for double labelling. For more examples, please refer to the Jackson Catalogue or contact Stratech Scientific.
The success of these experimental designs is unpredictable and may require some empirical manipulations. Trying different concentrations of reagents in each step or switching the labelling sequence of the two antigens may sometimes influence the outcome. Blocking with an appropriate normal serum between certain steps may also help to reduce background. To avoid release of the blocking Fab antibodies by labelled secondary antibodies, the tissue or cells may be lightly fixed after the blocking step with a fixative, such a glutaraldehyde, provided that this fixation does not severely affect antigenicity of the second antigen to be labelled.

Figure 1. Example A, use of conjugated Fab fragments for labelling and blocking. Steps: 1 Incubate with the first, primary antibody. 2 Incubate with an excess of Probe 1-conjugated Fab antibody against   the host species of the primary antibody. 3 Proceed with the labelling of the second antigen as usual Caution: This protocol may be subject to the following problems: 1) Example A may require a higher concentration of conjugated Fab to achieve effective blocking of the first primary antibody. If this results in a higher background, try a lower concentration of the conjugated Fab followed by further blocking with unconjugated Fab. 2) If small aggregates of conjugated Fab are present and they bind to the first primary antibody, they may also act as divalent or polyvalent molecules and capture some of the second primary antibody, which could result in overlapping detection of antigens.

Figure 2. Example B, Use of unconjugated Fab fragments to convert the first, primary antibody into a different species Steps: 1 Incubate with the first, primary antibody. 2 Incubate with an excess of unconjugated Fab antibody directed   against the host species of the primary antibody. 3 Incubate with Probe 1-conjugated tertiary antibody [an anti-IgG   (H+L) or an anti-F(ab’)2] directed against the host species of the   Fab antibody. It is very important that the tertiary antibody does   not recognise the host species of the either the primary antibodies or   the second, secondary antibody. 4 Incubate with the second, primary antibody. 5 Incubate with Probe II-conjugated to the second, secondary   antibody (that does not recognise the host species of either the Fab antibody or the tertiary antibody).

Legend

By Jaquie Finn.
Jaquie worked for 10 years in the field of molecular biology before moving into bioinformatics. She is currently working at Stratech Scientific. Some of this article was also written by Dr W. Stegeman and Dr C. Ko, Jackson ImmunoResearch Laboratories.