Monovalent Fab fragments of affinity-purified, secondary antibodies are offered to cover (block) the surface of immunoglobulins for double labeling primary antibodies from the same host species, or to block endogenous immunoglobulins in tissue sections or on cell surfaces. They can be used for these purposes because each Fab fragment has only a single antigen binding site (i.e. they are monovalent).

In contrast, divalent antibodies (whole IgG and F(ab')₂ fragments) have two antigen binding sites. After labeling the first primary antibody, some antigen binding sites on the first secondary antibody may remain open which could capture the second primary antibody introduced in a subsequent step. Consequently, it will appear as overlapping labeling, even though there may not be overlapping antigens. Therefore, divalent antibodies should not be used for blocking or for double labeling two primary antibodies from the same species. 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 are not necessary when primary antibodies from the same host species are different classes of immunoglobulins, such as IgG and IgM, or different subclasses of IgG, such as Mouse IgG1 and Mouse IgG2a. In these cases, it is much easier and more effective to use class-specific or subclass-specific antibodies, respectively, to distinguish between the two primary antibodies.

To block endogenous immunoglobulins on cells or tissue sections, incubate with an excess (10-20 ug/ml) of unconjugated Fab antibody just after blocking with normal serum. To avoid a possible displacement of the Fab antibody by the labeled secondary antibody, a light post-fixation may be necessary, provided that it does not affect antigenicity of the target proteins. Fab antibodies are not effective for blocking immunoglobulins in Western blotting or ELISA applications.

Important note: The monovalent Fab fragments of secondary antibodies offered here have not been adsorbed to remove cross-reactivities to other species. Therefore, if the cells/tissues under study have endogenous immunoglobulins, using Example A or B may create background. In this case Example C should be used.

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 unconjugated primary antibodies from the same host species. The success of these experimental designs will require some empirical manipulations. In each case it is important to label the less abundant primary antibody first. Trying different concentrations of reagents in each step or switching the labeling sequence of the two antigens may influence the outcome. Blocking with an appropriate normal serum between certain steps may also help to reduce background. To avoid a possible displacement of the Fab antibody by the labeled secondary antibody, a light post-fixation may be necessary, provided that it does not affect antigenicity of the target proteins.

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 it is necessary to incubate with the labeled primary antibody first, blocking with a monovalent Fab antibody is required to prevent the secondary antibody used in a subsequent step from binding to the labeled primary antibody.

Click on one of the examples below to view the protocol: