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Reviewed July 5, 2021
The F(ab) fragment is an antibody structure that still binds to antigens but is monovalent with no Fc portion. An antibody digested by the enzyme papain yields two F(ab) fragments of about 50 kDa each and an Fc fragment.
In contrast, F(ab')2 fragment antibodies are generated by pepsin digestion of whole IgG antibodies (See below IgG structure) to remove most of the Fc region while leaving intact some of the hinge region. F(ab')2 fragments have two antigen-binding F(ab) portions linked together by disulfide bonds, and therefore are divalent with a molecular weight of about 110 kDa.
Figure legend: The light chain (LH) folds into a variable domain (VL) and a constant domain (CL) whereas the heavy chain is composed of one variable domain (VH) and three (IgG and IgA or four constant domains (IgE).
Monovalent antibody fragments (F(ab) fragments) are powerful tools to block background from primary antibody binding and in double staining experiments.
F(ab) fragments are used to block endogenous immunoglobulins on cells, tissues and exposed immunoglobulins in multiple labeling experiments using primary antibodies from the same species.
After the blocking step with normal serum, we recommend incubating F(ab) fragments in excess to block endogenous immunoglobulins in IHC. These antibodies are not recommended for blocking immunoglobulins in WB and ELISA.
Divalent antibody fragments (F(ab')2 fragments) are smaller than whole IgG molecules and enable a better penetration into tissue thus faciliting better antigen recognition in IHC. The use of F(ab')2 fragments also avoids unspecific binding to Fc receptor on live cells or to Protein A/G.
F(ab')2 fragments are not recommended for blocking since they have two binding sites that are available to capture the primary antibody introduced subsequently. However, as opposed to F(ab) fragments, F(ab')2 fragments can both bind and precipitate antigens thanks to their two binding sites. We recommend using normal serum with these antibodies to prevent the binding to Fc receptors.