Nonspecific binding of Clostridium difficile toxin A to murine immunoglobulins occurs via the fab component

Clostridium difficile toxin A binds nonspecifically to a mouse monoclonal antibody (MAb) immunoglobulin G3 lambda chain [IgG3(lambda)], through the Fab component. This binding, which is retained even after boiling the MAb, is temperature dependent, with more toxin bound at 4 than 37 degrees C (P = 0.0024). The nonspecific binding was decreased by incubation of the IgG3 lambda MAb with alpha- or beta-galactosidase (P = 0.0001 and 0.029, respectively), indicating that toxin A binds to a carbohydrate moiety on the Fab. However, binding was not blocked by the Bandeiraea simplicifolia lectin BS-1, indicating that a terminal alpha-galactose may not be involved. Binding was also not affected by competitive assays with Lewis X antigen. The dependence on carbohydrate moieties in nonspecific binding was also shown for two other MAbs, IgA(kappa) and IgM(lambda), with demonstration of a significant reduction in binding with alpha-galactosidase (P = 0.0001 and 0.0002, respectively) but not beta-galactosidase (P = 0.27 and 0.25, respectively).