Pepsin Digestion of Antibodies to Produce Functional Antigen-Binding Fragments (Fab): A Scientific Fantasy?

A pepsin antibody fragmentation procedure was trialed and optimised. The procedure used was a modified version of that described in Current Protocols in Immunology. Pilot digestions using a range of pepsin concentrations and incubation times were completed at pH 4.0 and pH 4.5. The digestion products were examined by Phastgel electrophoresis, and the combination of pepsin concentration, incubation time and pH that provided the most efficient digestion of the antibody into dimeric antigen-binding fragments (F(ab')2) was then used in the large-scale digestion of the antibody. Monomeric antigen binding fragments (Fab) were obtained through reduction of F(ab')2 using 2-mercaptoethanol. In this study Fab from a mouse polyclonal anti-ricin antibody and a sheep polyclonal anti-Bacillus anthracis antibody generated with the optimised procedure were comparable to the whole molecule when used as the capture antibody in an ELISA. Nevertheless, it was noted that, in spite of meticulous optimisation of the digestion process it can be fraught with problems.