Improved method for pepsinolysis of mouse IgG(1) molecules to F(ab')(2) fragments

Pepsinolysis of immunoglobulin (IgG) to yield F(ab')(2) fragments has been utilized for over 40 years, but the most common subclass of mouse immunoglobulin, IgG(1), is resistant to pepsin cleavage. We show here that this resistance is due to N-linked glycosylation. Deglycosylation by peptide: N-glycosidase F (PNGase F) improves pepsinolysis to generate F(ab')(2) fragments for mouse and rat monoclonal IgG(1) and, in some cases, IgG(2b). This effect was observed for antibodies derived from tissue cell culture or ascites fluid, as well as for polyclonal IgGs from mouse serum. As a result of this finding, the preparation of F(ab')(2) fragments from nearly all mouse IgG molecules should now be a robust procedure.