Identification of functionally relevant determinants on the complement component C3 with monoclonal antibodies.

The component C3 has a central function in both pathways of complement activation. Cleavage of C3 generates the fragments C3a and C3b, which have the capacity to trigger cellular functions. C3b interacts with several other complement components. The obvious versatility of C3 depends on distinct functional sites of the molecule. However, the broad specificity of conventional antisera to multiple determinants complicates an exact identification of antigenic sites on the C3 molecule. We used monoclonal antibodies (Ab) as a tool to correlate individual functions of C3 with defined antigenic determinants. A series of eight different monoclonal antibodies was produced after immunization with purified guinea pig C3. The antibodies were tested by ELISA against guinea pig or human C3, the fragments C3a, C3b, C3c, and C3d, or the separated α- and β-chains of guinea pig C3. Their ability to agglutinate or to bind to EA-C3b or EA-C3d cells and the capacity of mixtures of two antibodies to precipitate C3 was determined. In addition, we measured the effect of the antibodies on cleavage of native C3, on the uptake of 125l-labeled C3 by EAC142 cells, on the hemolytic function of cell-bound C3b, and on the ability of cell-bound C3b to react with radiolabeled B or H. Each antibody showed a typical reactivity pattern in these assays. Three informative examples are presented in more detail. Ab 103 reacted selectively with C3 and C3a but not C3b and inhibited the anaphylatoxic function of C3a in platelet release assays. Ab 111 reacted with C3, C3b, C3c, and the α-chain of C3 but not C3d or the β-chain; this antibody interferes with the hemolytic function of cell-bound C3b, and it strongly inhibited the binding of B but not of H. Ab 109 reacts with a determinant present on C3, C3b, and C3d but not C3c. In the presence of Ab 109 an increased uptake of C3 occurred. It strongly inhibited H but not B uptake, which indicated again separate C3b-sites for these components. Ab 109 increased the hemolytic activity of cell-bound C3b by protection against H; it converted trypsin-generated sheep E-C3b, a nonactivator of the alternative pathway (APC), into an APC-activator. Reagents with specificity for single determinants therefore apparently make it possible to identify and to discern functionally relevant sites of the C3 molecule, and they permit a selective manipulation of a given biologic function of C3.