Antibody Fragmentation

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. The Fc fragment is digested into small peptides. Fab fragments are generated by cleavage of IgG with papain instead of pepsin. Papain cleaves IgG 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. The fragments can be purified by gel filtration, ion exchange, or affinity chromatography. The properties of these fragments are summarized in Table A.

Fab and F (ab′) 2 antibody fragments are used in assay systems where the presence of the Fc region may cause problems. In tissues such as lymph nodes or spleen, or in peripheral blood preparations, cells with Fc receptors (macrophages, monocytes, B lymphocytes, and natural killer cells) are present which can bind the Fc region of intact antibodies, causing background staining in areas that do not contain the target antigen. Use of F (ab′) 2 or Fab fragments ensures that the antibodies are binding to the antigen and not to Fc receptors.1 These fragments may also be desirable for staining cell preparations in the presence of plasma, because they are not able to bind complement, which could lyse the cells. F (ab′) 2, and to a greater extent Fab, fragments allow more exact localization of the target antigen, i.e. in staining tissue for electron microscopy. The divalency of the F(ab′) 2 fragment enables it to cross-link antigens, allowing use for precipitation assays, cellular aggregation via surface antigens,2 or rosetting assays.3

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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
P7012 Pepsin from porcine gastric mucosa lyophilized powder, ≥2,500 units/mg protein (E1%/280)