Fab fragment antibodies are generated by papain digestion and subsequent purification to yield the approximately 50 kDa monovalent Fab antibody portion of the molecule. These fragments lack the Fc portion of the molecule and will not interact with either in vitro or cellular Fc binding mechanisms. The relatively low molecular weight of these reagents maximizes permeability for in situ studies.
Fab fragment antibodies are useful in experiments where two primary antibodies from the same host species are used, such as two IgG-type mouse monoclonal antibodies. A conjugated Fab fragment anti-mouse IgG is applied, which binds to the surface of the mouse IgG antibody molecule, completely masking all sites for anti-mouse IgG binding. In addition, because of the Fab fragment antibody’s monovalent properties, it is not able to bind any additional mouse IgG antibody if it were reintroduced into the system. At this point, the second primary antibody, also mouse IgG, is applied and subsequently detected with a second conjugated anti-mouse IgG. The masking properties of the conjugated Fab fragment antibody used to bind the first primary antibody shield the first mouse IgG from detection by the second mouse antibody.
F(ab')2 Fragment Secondary Antibodies
F(ab')2 fragment secondary antibodies are generated by pepsin digestion of whole IgG antibodies to remove most of the Fc region while leaving intact the hinge region. F(ab')2 fragments have two antigen-binding F(ab) portions linked together by disulfide bonds, and therefore have a molecular weight of about 110 kDa.
F(ab')2 fragment antibodies eliminate non-specific binding between the Fc portions of antibodies and the Fc receptors on cells. When working with tissues or cells that have Fc receptors (spleen, blood, hematopoietic cells, leukocytes, etc.), choose an F(ab')2 fragment to eliminate non-specific binding to Fc receptors. F(ab')2 fragment conjugated secondary antibodies are ideal for flow cytometry, immunohistochemistry, and immunofluorescence.
