Measurement of complement activation in rabbit plasma or serum using monoclonal antibodies against C5a

Abstract

Eight murine monoclonal antibodies (MoAb) raised against the major zymosan-induced chemotactic factor in rabbit serum were found to neutralize the chemotactic activity induced by lipopolysaccharides (LPS) and antigen-antibody complexes. A 15 kDa antigen was identified in plasma incubated with LPS by immunoblot analysis with MoAb. This is similar to the molecular weight of the major zymosan-induced chemotactic factor. Both the generation of this 15 kDa antigen and chemotactic activity were abrogated in a heat-inactivated plasma. A cross-reaction to human C5a was demonstrated for three MoAb (5H8B9, 4B1C11, and 2A5E3) in an indirect enzyme-linked immunosorbent assay (ELISA) of partially purified C5a and by the isolation of zymosan-induced chemotactic activity by affinity chromatography. MoAb 5H8B9 and 4B1C11 were able to neutralize the chemotactic activity in human zymosan-activated serum. MoAb 2A5E3 was able to bind 125I-labelled human C5a des Arg. We conclude that these MoAb are directed against rabbit C5a. MoAb 5B2C5 and 2B1A2, which are directed to different antigenic binding sites on C5a, may be applied in a sandwich ELISA for the detection and quantification of C5a des Arg in rabbit serum or plasma. The sandwich ELISA can be performed directly on serum or plasma samples without having to precipitate native C5. Complement activation is demonstrated by measuring the increased generation of C5a des Arg in rabbit plasma or serum activated with LPS, zymosan, antigen-antibody complexes, or cobra venom factor.