Hemolytic plaque inhibition: the physical chemical limits on its use as an affinity assay

An analysis of the fundamental physical chemical limits of hemolytic plaque inhibition as a method for obtaining thermodynamic and kinetic information is presented. It is shown that inhibition curve characteristics will be sensitive functions of reaction affinities only when the antibody inhibitor and antibody red blood cell reaction mechanisms are related in certain ways. It is further shown that conditions which are most sensititive to IgG affinity changes will generally not be the best for detecting changes in IgM affinity. The apparently conflicting reports on IgM maturation are completely explicable in terms of experimental requirements imposed by physical chemical characteristics of the reaction. Experiments in which maturation is observed are found to conform to the most sensitive conditions of the assay, whereas those in which it is not observed are found to conform to relatively insensitive conditions, and would therefore be capable of registering changes only when affinity shifts are large.