N-GLYCANASE TREATMENT OF F(AB')(2) DERIVED FROM ASYMMETRIC MURINE IGG(3) MAB DETERMINES THE ACQUISITION OF PRECIPITATING ACTIVITY

The aim of this study was to analyse four anti-DNP asymmetrically glyc osylated monoclonal IgG3 antibodies (194/2, 194/5, 194/6 and 194/12) b efore and after carbohydrate manipulation. Microheterogeneity in the c omposition of the carbohydrate moiety involved in Fab' glycosylation w as detected using lectins. Additional O-glycosidic carbohydrate chains were detected within the Fc region of two monoclonal antibodies. Fab' glycosylation produced a difference in the binding constants (K-a) in each paratope of two orders of magnitude, as determined by means of p rimary ligand-antibody interaction. The difference in binding affinity and the importance of Fc-Fc interaction was evidenced by a lack of BS A-DNP precipitation by the F(ab')(2) fragments. The oxidation of the a ntibodies with sodium periodate caused the disappearance of the low af finity binding site as determined by fluorescence quenching. Furthermo re, the enzymatic removal of the carbohydrate with N-glycanase determi ned the acquisition of precipitating activity by the F(ab')(2) fragmen ts.