Relationship between antibody affinity and hemolytic plaque diameter—II: Maturation of primary immune responses to DNP and SRBC

The primary immune responses of C3H mice to the model monodeterminant antigen DNP2 and the multideterminant antigen SRBC were studied in order to determine the relationship between relative antibody affinity and the hemolytic plaque diameter of individual antibody-secreting cells during maturation of the primary immune responses. Competitive inhibition PFC assays, employing either DNP-lysine or SRBC stromata, were used to assess relative antibody affinity and the degree of heterogeneity. Both immune responses exhibited typical maturational features with a progressive increase in mean antibody affinity and a progressive decrease in the heterogeneity of antibody affinities. The mean diameter of anti-DNP plaques increased during maturation and high affinity anti-DNP antibody produced larger plaques than low affinity antibody. However, the mean plaque diamter of anti SRBC plaques decreased during maturation. This discrepancy was explored by analysis of the distribution of PFC sizes and the capacity of large vs small plaques to be neutralized by competing antigen. The anti-SRBC response was characterized by a significantly greater frequency of small plaques during maturation as compared to the anti-DNP response. These new small plaques were less susceptible to inhibition than the larger plaques, a result consistent with the progressive emergence of PFC producing low affinity antibody. This study suggests that immunization with a complex multideterminant antigen such as SRBC may significantly influence the distribution of plaque sizes. Whereas competitive inhibition PFC analyses may provide a relative measure of antibody affinity and the clonal distribution of antibody affinities in responses to both monodeterminant and multideterminant antigens, analysis of maturation by reference to plaque diameter per se wean be erroneous, owing perhaps to the appearance of subpopulations of PFC synthesizing low affinity antibody or antibody directed to less dense determinants.