IgG pair formation on one antigenic molecule is the main mechanism of synergy between antibodies in complement-mediated lysis.

Two rat IgG2b monoclonal antibodies bound to different epitopes on a offgle RT1Aa antigen molecule synergize in their induction of complement-mediated lysis of the red cells. The mechanism of this synergistic action has been investigated by determining the C1q:IgG relationship on red cells using the fluorescence-activated cell sorter and making use of the considerable variation in antigen density between individual red cells. With a synergistic pair of antibodies, the C1q:IgG ratio was approximately 1 and independent of antibody density per cell, indicating that the antibody molecules are present as closely associated pairs, each pair binding two C1q molecules and thus forming a cyclic tetramer. When one of the antibodies was used alone, the number of C1q molecules bound relative to the amount of antibody was higher than expected from theoretical considerations based on a presumed random distribution of antigen-antibody complex. This result can be explained if there is some mobility of the complex within the membrane. The interpretation of the results gives strong support to the hypothesis that C1q must bind bivalently to rat IgG2b complexes.