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
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.