Characterization of the interaction between murine tumour necrosis factor and monoclonal antibodies

Characterization of the interaction between murine tumour necrosis factor and monoclonal antibodies.Ameloot P, Fiers W, Buurman WA, de Baetselier P, Brouckaert P.Department of Molecular Biology, Unit of Molecular Pathophysiology and Experimental Therapy, Flanders Interuniversity Institute for Biotechnology and Ghent University, K.L. Ledeganckstraat 35, 9000, Ghent, Belgium.We characterized the interaction between murine TNF (mTNF) and the neutralizing monoclonal antibodies TN3 and 1F3F3. The epitopes were localized by comparing the detection efficiency for a panel of TNF chimaeric proteins and site-specific muteins in ELISA. Mutation of mTNF amino acid Q131 inhibited the interaction with 1F3F3, whereas mutation of D71/Y72 inhibited the binding to TN3. As D71/Y72 are located in an exposed loop near the TNF intersubunit groove, binding of TN3 promoted the dissociation and/or interfered with the reassociation of subunits into mTNF trimers. 1F3F3, on the other hand, prevented the spontaneous dissociation of bound (hetero)trimeric mTNF.