Rockland’s experience in working with antibody enzymatic cleavage allows us to process immunoglobulins into their constituent components. In some assays, it is preferable to use only the antigen-binding (Fab) portion of the antibody to prevent the constant (Fc) portion from binding to cell surface receptors. For these applications, antibodies can be enzymatically digested using the enzyme papain to produce a Fab fragment and/or Fc fragment of the antibody or the enzyme pepsin to produce a F(ab')2 fragment of the antibody. While Rockland offers secondary antibodies as Fab fragments and F(ab’)2 fragments, primary antibodies are digested into fragments as a custom service.
F(ab')2 DIGESTION ( up to 100 mg of purified IgG)
Fab Digestion (up to 100 mg of purified IgG)
TIME COURSE STUDY - for papain or pepsin digests including analysis by SDS-PAGE
Some immunoassays require an antibody fragment for optimal activity. These immunoglobulin fragments are typically Fc, Fab or F(ab’)2 (see figure 1). Digestion of IgG with pepsin produces the F(ab’)2 fragment. The F(ab’)2 fragment contains 2 covalently linked Fab region while leaving intact some of the hinge region. F(ab')2 fragments have two antigen-binding Fab domains and are divalent with a molecular weight of approximately 110 kDa. Digestion of an IgG with papain produces three products: one Fc fragment and two identical Fab fragments. The Fab fragment is a 50 kDa disulfide bonded antibody fragment that retains its antigen binding activity but lacks the Fc portion of the IgG. The Fc fragment is composed of parts of two heavy chains. It has no antigen-binding ability, but does contribute to a number of effector functions.
Rockland’s antibody digest services include dialysis, digestion and purification of the proteolytic fragments. Digestion is by pepsin or papain to produce F(ab’)2, Fab or Fc fragments as described above. Typical projects start with up to 100 mg of an IgG antibody, but larger scale digests are routinely performed depending on need and application.
For custom antibody fragmentation, a digestion time course study is recommended to optimize antibody-specific conditions and efficiently manage precious primary antibody. Because each IgG is different, optimal digestion conditions for one antibody may not be expected for all other antibodies of that species or subclass. A pilot experiment should always be carried out when a new antibody is to be digested with papain or pepsin. In a time course study a sample of antibody is digested with the enzyme for varying amounts of time to determine the optimal conditions for digestion of the antibody in larger scale. Figure 2 shows an example of a coomassie stained gel for a typical pepsin F(ab’)2 time course study.
Related Custom Services
More information on Rockland’s adherence to QSR / cGMP quality standards (CFR:21H part 820) can be found at The Rockland Advantage. Our rigorous quality control testing procedures include Enzyme-linked immunosorbent assay (ELISA), Western Blot, Immunofluorescence microscopy, fresh and frozen section Immunohistochemistry, PEFF Immunohistochemistry, High-performance liquid chromatography (FPLC and HPLC), Size Exclusion Chromatography (SEC), antibody conjugation and fragmentation, bioburden assays, and both one dimensional and two dimensional (1D and 2D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Rockland Immunochemicals Inc.Limerick, PA 19468E-mail: email@example.comPhone: 800.656.7625