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| Fragment Format-1: | Fab | Fragment Format-2: | F(ab')2 |
|---|---|---|---|
| Enzyme-1: | Papain | Enzyme-2: | Pepsin |
| Method: | Enzymatic Digestion | Starting Material: | Whole IgG |
| High Light: | Enzymatic Digestion Antibody Fragmentation,Antibody Modifications Conjugation Fragmentation |
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Introduction
Antibody fragments are functional regions of whole immunoglobulin consisting of the antigen binding fragment (Fab) and the crystallizable fragment (Fc). Fabs may be used to bind their target antigen without directing an immune response, resulting in extremely controlled and pointed targeting. Removal of the crystallizable or constant fragment (Fc) region also reduces potential unwanted non-specific interactions during biochemical assays such as immunohistochemistry. Additionally, antibody fragments can reach 1/3 the size of whole IgG (50kDa vs 150kDa), making them useful in applications which require smaller targeting molecules to penetrate tissue or reduce steric hindrance.
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Antibody Fragment Format |
Digestion Enzyme |
Service Items |
|---|---|---|
| Fab | papain | Fab Fragmentation |
| F(ab')2 | pepsin | F(ab')2 Fragmentation |
Papain digestion: Fab from IgG
Papain is a nonspecific, thiol-endopeptidase that has a sulfhydryl group in the active site, which must be in the reduced form for activity. When IgG molecules are incubated with papain in the presence of a reducing agent, one or more peptide bonds in the hinge region are split, producing three fragments of similar size: two Fab fragment and one Fc fragment (1). When Fc fragments are of interest, papain is the enzyme of choice because it yields an intact 50,000-dalton Fc fragment.
Pepsin is a nonspecific endopeptidase that is active only at acid pH. It is irreversibly denatured at neutral or alkaline pH. Digestion by the enzyme pepsin normally produces one F(ab')2 fragment and numerous small peptides of the Fc portion. The resulting F(ab')2 fragment is composed of two disulfide-connected Fab units. The Fc fragment is extensively degraded, and its small fragments can be separated from F(ab')2 by dialysis, gel filtration or ion exchange chromatography.
Advantages of Fab and F(ab')2 Fragmentation
