Immunoglobulins can be cleaved by proteolytic enzymes into functionally distinct subunits. Generally, fragments that bind antigens are of interest. Fab and F(ab′)2 fragments contain the antibody-binding portion of the molecule; the Fc fragment is generally only of interest if studying the crystal structure of antibodies. The Fab or F(ab′)2 portion of the antibody may be required in certain experiments. For instance, in mixed lymphocyte response (MLR) and cytolytic T lymphocyte (CTL) assays, the Fc portion may cause artifacts because it binds to cells bearing the Fc receptor. It has also been suggested that the Fc portion of the antibody may be more immunogenic in vivo (Hellstrom and Hellstrom, 1985). The Fc portion of IgG2a and IgG3 can invoke antibody-dependent cell-mediated cytotoxicity responses in vivo and in vitro.
The monoclonal antibodies routinely used for in vitro and in vivo experiments are from the mouse. Subclass-dependent susceptibility of mouse antibodies to proteolytic cleavage has been widely reported (Parham, 1986). The order of sensitivity to proteolytic enzymes has been found to be IgG2b>IgG3>IgG2a>IgG1. Not all antibodies, however, fall into this ranking; IgG2a can be extremely sensitive to the action of papain in the presence of cysteine. If such sensitivities are found, then the method in which cysteine is removed from the enzyme following preactivation is recommended (Parham et al., 1982).
It must be stressed that all monoclonal antibodies are different and, for this reason, standard conditions for fragmentation are not given for papain and pepsin digestion. A pilot experiment should always be performed to assess optimal conditions before carrying out a largescale fragmentation. Microdialysis (Overall, 1987) is recommended so that the conditions from small- to large-scale are identical.
IgG can be fragmented to yield monovalent Fab fragments using the proteolytic enzyme papain in the presence of the reducing agent cysteine. IgG subclasses (e.g., IgG1 and IgG2) have different susceptibilities to papain digestion. IgG1, IgG2a, and IgG3 can be digested to bivalent F(ab′)2 fragments using pepsin. Although IgG1 may be sensitive to pepsin degradation, a more stable digestion is achieved using papain in the absence of cysteine. Ficin is also useful for digestion of IgG1 to F(ab′)2 (Mariani et al., 1991). It is a thiol protease purified from fig latex. It requires activation with cysteine; the concentration of cysteine determines the extent of digestion. Generally, IgG2b cannot be fragmented to F(ab′)2. Prior to fragmentation, a pilot experiment should be undertaken to determine individual conditions for fragmentation of each antibody. Separation of fragments can be carried out very efficiently by ion-exchange (IEX) chromatography on DE52; other methods for separation also available at Bio-Synthesis.
Pepsin cleavage of mouse IgG subclasses results in two sites of proteolytic sensitivity, one on either side of the inter–heavy chain disulfide bonds as suggested by Parham (1983). Primary cleavage may occur at a site on the COOH-terminal side of the bridges, giving F(ab′)2, followed by secondary cleavage on the NH2-terminal side, giving Fab′. In the case of IgG2b, the relative sensitivities of these two sites is reversed. Because the heavy chains of IgG2b are asymmetrically glycosylated and the products of pepsin digestion of this subclass are Fab and Fab/c, F(ab′)2 has a molecular weight close to that of Fab/c. F(ab′)2, however, has rarely been reported to be obtained from IgG2b, and therefore is not a feasible product of this subclass.
Critical notes for conducting antibody fragmentation
Because each monoclonal IgG is different, if one antibody of a particular subclass is cleaved under a certain set of conditions, it cannot be expected that all other antibodies of that subclass will behave in the same manner. A pilot experiment should always be carried out when a new antibody is to be fragmented. It is probably better to choose a reaction time that will leave some antibody unfragmented rather than risk damaging the antibody-binding site by excessive exposure to proteolytic enzymes. Intact antibody can be easily removed from the fragmentation mixture by passage through a protein A–Sepharose column. For those antibodies that do not bind well to protein A, protein G–Sepharose or IEX chromatography are reasonable alternatives.
All Bio-Synthesis antibody fragmentation services includes pilot study to ensure optimize antibody activity for your experiment.