The time development of direct hemolytic plaques: implications for the binding of IgM to cell surface haptens

We studied the time development of direct hemolytic plaques in thin layers containing either sheep red blood cells (SRBC) directly haptenated (DH) with trinitrophenyl, or SRBC indirectly haptenated (IH) with dinitrophenyl coupled to human serum albumin. The DH-SRBC tend to be sparsely haptenated while the IH-SRBC tend to have very high local hapten densities. We observed marked differences in the growth of plaques for the two differently haptenated SRBC. Plots of the plaque radius squared vs time show that the slope of those curves that developed in a lawn of DH-SRBC tended to be constant while the slope of those curves that developed in a lawn of IH-SRBC tended to decrease with time. These results are what is predicted from theory if: IgM binds to DH-SRBC through attachments that rapidly dissociate, if IgM binds to IH-SRBC through attachments that very slowly dissociate, and if (3) both types of bound IgM can fix and activate complement.