We offer the following Fab fragments (see Figure 1) of affinity-purified, secondary antibodies to researchers who wish to sterically cover the surface of immunoglobulins for double labeling primary antibodies from the same host species, or to block endogenous immunoglobulins on cell surfaces or in 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 labeling 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, respectively, 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. Consequently, they might contribute to some degree of background staining for certain applications using Examples A or B. If this is a problem, Example C would be preferable.

Detection of two unlabeled primary antibodies from the same host species

The following examples show some of the possible protocols used for double labeling two unlabeled primary antibodies from the same host species. 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 labeling 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.

Please click on one of the examples below to view the protocol:

Detection of one unlabeled and one or more labeled primary antibodies from the same host species

It is important to incubate with the unlabeled primary antibody first.  This will obviate the need for blocking with a monovalent Fab antibody.  If, for some reason, it is necessary to incubate with the labeled primary antibody first, it is then necessary to block with a monovalent Fab fragment antibody, so that the secondary antibody used in a following step will not bind to the labeled primary antibody.

After the unlabeled primary antibody is detected with a labeled secondary antibody, any remaining IgG binding sites on the secondary antibody must be blocked before adding the labeled primary antibody.  This is readily done by incubating with a source of IgG, such as normal serum, gamma globulin, or purified IgG,  from the same species as the primary antibody.  An abbreviated protocol is given below.