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Antibody Fragmentation Services


Antibody Fragmentation Services

 

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.

 

 

Custom Service

Part number

F(ab')2 DIGESTION ( up to 100 mg of purified IgG)

CUST07 

Fab Digestion (up to 100 mg of purified IgG)

CUST29 

TIME COURSE STUDY - for papain or pepsin digests including analysis by SDS-PAGE

CUST59             

 

 

 

 

 

 

 

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Immunoglobulin Fab and F(ab’)2 fragments

 

 

Schematic representation of IgG fragments

Figure 1.  Schematic representation of IgG fragments produced from pepsin or papain cleavage.  “H” identifies the immunoglobulin heavy chain and “L” identifies the immunoglobulin light chain.  Disulfide bonds are indicated by black lines connecting the chains.

 

 

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.

 


 

Antibody Enzyme Digest (Pepsin or Papain) Services

 

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.

 

Recommended Services

  • For pepsin digestion of IgG to produce F(ab’)2 fragment primary antibody order code CUST07.
  • For papain digestion of IgG to produce Fab fragment primary antibody order code CUST29.

  


 

Digest Time Course Study


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.

 

 

time course study fab2

Figure 2. Digest fragments of IgG after limited digestion by the enzyme pepsin in a time course study. The IgG and the resultant F(ab’)2 fragment are labeled on the right.  

 

 

Recommended Services

 

  • For time course studies of IgG to determine optimal digest conditions order code CUST59

  


 

 

Related Custom Services

 

 


 

The Rockland  Advantage  

 

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).

 

 

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