Proteolytic Antibody Fragmentation

Fab and F (ab)2 antibody fragments are used in assay systems where the presence of the Fc region may cause problems. In these cases it is preferable to use only the antigen binding (Fab) portion of an antibody.

Papain is used routinely for the preparation of Fab fragments from IgG. IgM may also be digested with papain resulting in high yields of homogeneous Fab preparations. It cleaves above the hinge region containing the disulfide bonds that join the heavy chains, but below the site of the disulfide bond between the light chain and heavy chain. This generates two separate monovalent (containing a single antibody binding site) Fab fragments and an intact Fc fragment.

Pepsin is commonly used in the preparation of F(ab)2 fragments from antibodies. In some assays it is preferable to use only the antigen binding (Fab) portion of the antibody. For these applications, antibodies may be enzymatically digested to produce either an Fab or an F(ab)2 fragment of the antibody. To produce an F(ab)2 fragment IgG is digested with pepsin, which cleaves the heavy chains near the hinge region. One or more of the disulfide bonds that join the heavy chains in the hinge region are preserved, so the two Fab regions of the antibody remain joined together, yielding a divalent molecule (containing two antibody binding sites), hence the designation F(ab)2. The light chains remain intact and attached to the heavy chain.

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07298 FabRICATOR® for cleaving 2 mg IgG, 2000 U/vial
P4762 Papain from papaya latex lyophilized powder, ≥10 units/mg protein
P3125 Papain from papaya latex buffered aqueous suspension, 2× Crystallized, ≥16 units/mg protein
P6887 Pepsin from porcine gastric mucosa lyophilized powder, 3,200-4,500 units/mg protein