Antibodies papain digestion

Figure 20.11 Papain digestion of IgG antibodies primarily results in cleavage in the hinge region above the interchain disulfides. This produces two heavy-light chain pairs, called Fab fragments, each containing one antigen binding site. The Fc region normally can be recovered intact.

Moorehouse K.G., Nashabeh W., Deveney J., Bjork N.S., Mulkerrin M.G., and Ryskamp T. (1997), Validation of an HPLC method for the analysis of the charge heterogeneity of the recombinant monoclonal antibody IDEC-C2B8 after papain digestion, J. Pharm. Biomed. Anal. 16, 593-603. 

Fig. 2. Papain digestion of an antibody molecule yields two univalent Fab fragments and an Fc fragment whereas pepsin digestion yields a bivalent F(ab )2 fragment.

Figure 32.3. The structure of the antibody molecule. All immunoglobulins consist of a basic subunit made up of four polypeptide chains (two light and two heavy) bound together by disulfide bonds. The variable region of the molecule contains the antigen binding site. Papain digestion yields a constant fragment (Fc) and variable fragments (Fab).

Fig. 2. Immunoelectrophoresis of undigested goat IgG (upper well), and of Fab and Fc from papain digestion of goat IgG (lower well), showing the electrophoretic difference and the reaction of nonidentity between the Fab and the Fc fragments. The slot contains antibody to goat IgG.

Fig. 3. Immunoelectrophoresis of separated Fab (upper three wells) and Fc (lower three wells), foHowing DEAE chromatography of a papain digest of goat IgG. The slots contain antibody to goat IgG.

Fig.l. Electroblots of chymotrypsin and papain digestion fragments, resolved by a 15% SDS elecrophoresis. Fixation of Zn. Ch)nnotrypsin digestion (1, 3), papain digestion (2, 4), low molecular mass standards (5). Autoradiograms after incubation with Zn (3, 4,5). Immunoblots after incubation with monoclonal antibody CI9 (1,2). 

Figure 5.35 Size-excisuion separation of Fat and fragments of a monoclonal antibody (mAb) after digestion with papain. Chromatographic conditions see Figure 5.34 injection volume 1 pL sample A 5mg/mL mAb and sample B papain digest peaks (1) mAb, (2) F t, and (3) F. ...

A family of 100 hybridoma antibodies can typically provide 20 tight binders and these need to be assayed for catalysis. At this stage in the production of an abzyme, the benefit of a sensitive, direct screen for product formation comes into its own. Following identification of a successful catalyst, the antibody is usually recloned to ensure purity and stabilization of the clone, then protein is produced in larger amount (—10 mg) and used for determination of the kinetics and mechanism of the catalysed process by classical biochemistry. Digestion of such protein with trypsin or papain provides fragment antibodies, Fabs, that contain only the attenuated upper limbs of the intact IgG (Fig. 1). It is these components that have been crystallized, in some... 

Papain is also used for partial digestion of antibodies. F(ab )2 are obtained when papain is free of cysteine. The interest of these fragments is their higher antigen avidity when compared to Fab fragments. Those F(ab )2... 

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