Activation of complement by different immunoglobulin heavy chain isotypes of the channel catfish ( Ictalurus punctatus)

Abstract

The ∼ 750,000 mol. wt tetrameric Ab population from catfish immunized with sheep erythrocytes (E) was purified and shown to sensitize E to the hemolytic activity of catfish serum complement. Catfish complement was heat-labile at 45°C and was hemolytically inactivated by both EGTA and EDTA. Hemolytic activity of EDTA treated serum was restored by the addition of both Ca + and Mg 2+ ions. In comparative assays, the CH 50 titers of catfish sera were similar to the CH 50 titers of human sera. Catfish complement was hemolytically active at incubation temps ranging from 3 to 40°C, which suggests that complement should be functional throughout the catfish's normal environmental temps. Human complement exhibited considerable cross-reactivity when assayed on catfish Ab sensitized E at 25°C; however, at 3°C, no hemolytic activity was observed. These findings suggest that there may be structural differences between the complement component(s) of these two systems. Mouse mAbs to the recently described H chain isotypes of catfish Ig were used to fractionate catfish anti-sheep E antisera. Each of the purified isotypes contained Ab which sensitized E to the hemolytic activity of catfish complement. Dose-response analysis of the average number of hemolytic sites per cell, as a function of relative Ab concn, predicts that the catfish tetramer conforms to the one-hit theory of immune hemolysis. Purified catfish anti-fluorescein Ab of each isotype was also reacted with fluorescein-labeled sheep E and the level of complement-mediated hemolysis determined. Dose-response analysis indicated that similar numbers of Ab molecules bound to haptenated E resulted in similar levels of complement-mediated lysis. Lastly, the effect of cell surface hapten density of target cells was examined. These studies, similar to those reported with human IgM, showed that the binding of the catfish tetramer to a cell surface hapten may not be sufficient to activate lytic complement.