Characterization of three monoclonal antibodies against C3 with selective specificities

We raised 15 mouse monoclonal antibodies against human C3 from two separate fusions. Mouse plasmacytoma cells were fused with spleen cells from mice immunized either with a mixture of C3, C3b and C3c, or with a mixture of C3dg and C3d. Three of the 15 monoclonals were characterized in detail. N-7A reacted with native C3 as well as C3b and C3c. Two other monoclonals, C-5G and G-3E, recognized neoantigenic determinants on C3c and C3dg, respectively. In ELISA, C-5G reacted with C3b and C3c but not with native C3 nor with C3dg; and on the other hand G-3E reacted only with C3dg. The selective specificities of these monoclonals were further confirmed in a binding assay to C3 fragments formed on cellular intermediates. C-5G bound exclusively to EC3b, and G-3E bound to EiC3b, EC3dg and EC3d. N-7A bound only very poorly to EC3b. C-5G inhibited both the haemolytic activity of C5 convertase and also the CR1-mediated rosette formation of B-enriched peripheral mononuclear cells with EC3b. G-3E inhibited the CR2-mediated EC3dg-rosette formation of Raji cells. These monoclonals, with selective specificities, can distinguish the state of activation and degradation of the C3 molecule.