Preparation of F(ab’)2 Fragments from Mouse IgG of Various Subclasses (1)
Peptic digestion of immunoglobulins has been widely used for the preparation of fragments of IgG antibodies that are bivalent but lack the Fc region. Four myeloma proteins These were LPC-1 (IgG2a); MOPC-21(IgG1); J606 (IgG3); and MPC-II (IgG2b) were also used for this study.
Peptic digestion of IgG1. The time course of peptic digestion of two IgG1 proteins: R16.7, an MA with anti-Ar activity (lanes 1-5) and an IgGl fraction of specifically purified A/J serum anti-Ar (lanes 6-9). The digestion of MA R16.7 yields a major product with a relative molecular weight of 108,000, a value which agrees well with the expected molecular weight of F(ab’) 2 fragments. The digestion appears to be nearly complete after 12 h. 70% of the total anti-Ar activity remained after 12 h of peptic digestion of MA R16.7; however, only 10% of the original activity was associated with molecules that still carried the Fc segment.
Peptic digestion of IgG2a. Digestion of both proteins, R10.8 (lanes 1-5) and R22.4 (lanes 6- 10), was virtually complete after 8 h. Each major product has a relative molecular weight, estimated from its mobility, of 108,000- 110,000. After 8 h of digestion, 29% and 25%, respectively, of the original Ar-binding activity remained in the two proteins, but nearly all of this activity was associated with molecules lacking an Fc segment. Thus, the digestion of the IgG2a proteins proceeded somewhat more rapidly than that of the two IgG1 proteins.
Peptic digestion of IgG2b. The 4 anti-Ar MA, R8.2, R9.3, R19.9, and R23.2, were found to be degraded virtually completely by pepsin within 30 min at pH 4.2 (37°C). Protein R8.2 was
digested for 15 min at pH 4.2 or at pH 4.5. At pH 4.5, 29% of the original anti-Ar activity remained but all of this activity was associated with molecules that contained an Fc fragment. After 15 min at pH 4.2, only 10% of the anti-Ar activity remained; again, all of this activity was associated with undegraded IgG.
Peptic digestion of lgG3. Of digestion of MA R64.8, within 15 min the band corresponding to IgG had disappeared; the major band had an M r of 110,000. As indicated in Table III, 59% of the original hapten-binding activity remained after 15 min of digestion and none of this activity was associated with molecules that possessed an Fc region, i.e., with undegraded IgG3. Protein J606 was more resistant to prolonged digestion than MA R64.8.
The results indicate that F(ab’)2 fragments can be obtained in moderate to excellent yield from mouse IgGI, IgG2a and IgG3 by digestion with pepsin however, IgG2b is rapidly degraded to small peptides.