Monovalent Fab Fragment Affinity-Purified Antibodies for Blocking and Double Labeling Primary Antibodies from the Same Host Species
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 Fab fragments have 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.
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.
Caution: Whole IgG and F(ab')2 fragments are divalent antibodies, with two antigen binding sites, therefore they cannot be used in the following protocols which specifically require Fab fragments.
Detection of two unlabeled primary antibodies from the same host species
Labeling with two unconjugated primary antibodies of the same host species.
A Detection method using conjugated Fab fragments and whole IgG secondary antibodies.
Labeling with two unconjugated primary antibodies of the same species.
A blocking method using unconjugated fab fragments to “change” the species of one antibody.
Labeling using two unconjugated primary antibodies of the same species after blocking for endogenous Ig.
A blocking method to allow use of two same species primary antibodies following blocking for endogenous Ig with unconjugated fab fragments.
- The monovalent Fab fragments have not been adsorbed to remove cross-reactivities to other species. If the experimental sample contains endogenous immunoglobulins Example C should be used. Example A or B could introduce background.
Detection of one unlabeled and one or more labeled primary antibodies from the same host species
Labeling using unconjugated and conjugated primary antibodies of the same host species.
A blocking method using unconjugated fab fragments to prevent detection of the conjugated primary by subsequent antibodies.
Labeling using conjugated and unconjugated primary antibodies of the same species.
A blocking method using whole Ig antibodies to prevent non-specific labeling.